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Punetha, Jaya, and Eric P. Hoffman. "Short Read (Next-Generation) Sequencing." Circulation: Cardiovascular Genetics 6, no. 4 (2013): 427–34. http://dx.doi.org/10.1161/circgenetics.113.000085.
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Rodrigue, Sébastien, Arne C. Materna, Sonia C. Timberlake, et al. "Unlocking Short Read Sequencing for Metagenomics." PLoS ONE 5, no. 7 (2010): e11840. http://dx.doi.org/10.1371/journal.pone.0011840.
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Jackman, Shaun D., and İnanç Birol. "Assembling genomes using short-read sequencing technology." Genome Biology 11, no. 1 (2010): 202. http://dx.doi.org/10.1186/gb-2010-11-1-202.
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Simon, Stacey A., Jixian Zhai, Raja Sekhar Nandety, et al. "Short-Read Sequencing Technologies for Transcriptional Analyses." Annual Review of Plant Biology 60, no. 1 (2009): 305–33. http://dx.doi.org/10.1146/annurev.arplant.043008.092032.
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Botton, Mariana R., Yao Yang, Erick R. Scott, Robert J. Desnick, and Stuart A. Scott. "Phased Haplotype Resolution of the SLC6A4 Promoter Using Long-Read Single Molecule Real-Time (SMRT) Sequencing." Genes 11, no. 11 (2020): 1333. http://dx.doi.org/10.3390/genes11111333.
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The SLC6A4 gene has been implicated in psychiatric disorder susceptibility and antidepressant response variability. The SLC6A4 promoter is defined by a variable number of homologous 20–24 bp repeats (5-HTTLPR), and long (L) and short (S) alleles are associated with higher and lower expression, respectively. However, this insertion/deletion variant is most informative when considered as a haplotype with the rs25531 and rs25532 variants. Therefore, we developed a long-read single molecule real-time (SMRT) sequencing method to interrogate the SLC6A4 promoter region. A total of 120 samples were subjected to SLC6A4 long-read SMRT sequencing, primarily selected based on available short-read sequencing data. Short-read genome sequencing from the 1000 Genomes (1KG) Project (~5X) and the Genetic Testing Reference Material Coordination Program (~45X), as well as high-depth short-read capture-based sequencing (~330X), could not identify the 5-HTTLPR short (S) allele, nor could short-read sequencing phase any identified variants. In contrast, long-read SMRT sequencing unambiguously identified the 5-HTTLPR short (S) allele (frequency of 0.467) and phased SLC6A4 promoter haplotypes. Additionally, discordant rs25531 genotypes were reviewed and determined to be short-read errors. Taken together, long-read SMRT sequencing is an innovative and robust method for phased resolution of the SLC6A4 promoter, which could enable more accurate pharmacogenetic testing for both research and clinical applications.
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Eisenstein, Michael. "Startups use short-read data to expand long-read sequencing market." Nature Biotechnology 33, no. 5 (2015): 433–35. http://dx.doi.org/10.1038/nbt0515-433.
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Peng, Mengfei, Morgan L. Davis, Meghan L. Bentz, et al. "Short-Read and Long-Read Whole Genome Sequencing for SARS-CoV-2 Variants Identification." Viruses 17, no. 4 (2025): 584. https://doi.org/10.3390/v17040584.
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Genomic surveillance of SARS-CoV-2 is crucial for detecting emerging variants and informing public health responses. Various sequencing technologies are used for whole genome sequencing of SARS-CoV-2. This cross-platform benchmark study applied established bioinformatics tools to assess and improve the performance of Illumina NovaSeq, Oxford Nanopore Technologies MinION, and Pacific Biosciences Sequel II sequencing platforms in identifying SARS-CoV-2 variants and lineage assignment. NovaSeq produced the highest number of reads and bases, depth of coverage, completeness of consensus genomes, stable mapping coverage across open reading frames in the genome, and consistent lineage assignments. The long-read sequencing platforms had lower yields, sequencing depth, and mapping coverage, limiting the number of qualified sequences for lineage assignment and variant identification. However, implementing proper quality controls on sequence data overcame these limitations and achieved consistent SARS-CoV-2 lineage assignments across all three sequencing platforms. The advancements in library preparation and technology for long-read sequencing are likely to enhance sequence quality and expand genome coverage, effectively addressing current limitations in genome analysis. By merging the unique advantages of both short- and long-read methods, we can significantly improve SARS-CoV-2 genomic surveillance and provide insights into sequencing strategies for other RNA viruses, pending further validation. This may lead to precise tracking of viral evolution and support public health policy decisions.
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Kumar, Ashwini, Sadiksha Adhikari, Matti Kankainen, and Caroline A. Heckman. "Comparison of Structural and Short Variants Detected by Linked-Read and Whole-Exome Sequencing in Multiple Myeloma." Cancers 13, no. 6 (2021): 1212. http://dx.doi.org/10.3390/cancers13061212.
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Linked-read sequencing was developed to aid the detection of large structural variants (SVs) from short-read sequencing efforts. We performed a systematic evaluation to determine if linked-read exome sequencing provides more comprehensive and clinically relevant information than whole-exome sequencing (WES) when applied to the same set of multiple myeloma patient samples. We report that linked-read sequencing detected a higher number of SVs (n = 18,455) than WES (n = 4065). However, linked-read predictions were dominated by inversions (92.4%), leading to poor detection of other types of SVs. In contrast, WES detected 56.3% deletions, 32.6% insertions, 6.7% translocations, 3.3% duplications and 1.2% inversions. Surprisingly, the quantitative performance assessment suggested a higher performance for WES (AUC = 0.791) compared to linked-read sequencing (AUC = 0.766) for detecting clinically validated cytogenetic alterations. We also found that linked-read sequencing detected more short variants (n = 704) compared to WES (n = 109). WES detected somatic mutations in all MM-related genes while linked-read sequencing failed to detect certain mutations. The comparison of somatic mutations detected using linked-read, WES and RNA-seq revealed that WES and RNA-seq detected more mutations than linked-read sequencing. These data indicate that WES outperforms and is more efficient than linked-read sequencing for detecting clinically relevant SVs and MM-specific short variants.
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Eisenstein, Michael. "Innovative technologies crowd the short-read sequencing market." Nature 614, no. 7949 (2023): 798–800. http://dx.doi.org/10.1038/d41586-023-00512-4.
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Yu, Xiaoling, Wenqian Jiang, Xinhui Huang, Jun Lin, Hanhui Ye, and Baorong Liu. "rRNA Analysis Based on Long-Read High-Throughput Sequencing Reveals a More Accurate Diagnostic for the Bacterial Infection of Ascites." BioMed Research International 2021 (November 17, 2021): 1–8. http://dx.doi.org/10.1155/2021/6287280.
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Traditional pathogenic diagnosis presents defects such as a low positivity rate, inability to identify uncultured microorganisms, and time-consuming nature. Clinical metagenomics next-generation sequencing can be used to detect any pathogen, compensating for the shortcomings of traditional pathogenic diagnosis. We report third-generation long-read sequencing results and second-generation short-read sequencing results for ascitic fluid from a patient with liver ascites and compared the two types of sequencing results with the results of traditional clinical microbial culture. The distribution of pathogenic microbial species revealed by the two types of sequencing results was quite different, and the third-generation sequencing results were consistent with the results of traditional microbial culture, which can effectively guide subsequent treatment. Short reads, the lack of amplification, and enrichment to amplify signals from trace pathogens, and host background noise may be the reasons for the high error in the second-generation short-read sequencing results. Therefore, we propose that long-read-based rRNA analysis technology is superior to the short-read shotgun-based metagenomics method in the identification of pathogenic bacteria.
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Thibodeau, My Linh, Kieran O’Neill, Katherine Dixon, et al. "Improved structural variant interpretation for hereditary cancer susceptibility using long-read sequencing." Genetics in Medicine 22, no. 11 (2020): 1892–97. http://dx.doi.org/10.1038/s41436-020-0880-8.
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Abstract Purpose Structural variants (SVs) may be an underestimated cause of hereditary cancer syndromes given the current limitations of short-read next-generation sequencing. Here we investigated the utility of long-read sequencing in resolving germline SVs in cancer susceptibility genes detected through short-read genome sequencing. Methods Known or suspected deleterious germline SVs were identified using Illumina genome sequencing across a cohort of 669 advanced cancer patients with paired tumor genome and transcriptome sequencing. Candidate SVs were subsequently assessed by Oxford Nanopore long-read sequencing. Results Nanopore sequencing confirmed eight simple pathogenic or likely pathogenic SVs, resolving three additional variants whose impact could not be fully elucidated through short-read sequencing. A recurrent sequencing artifact on chromosome 16p13 and one complex rearrangement on chromosome 5q35 were subsequently classified as likely benign, obviating the need for further clinical assessment. Variant configuration was further resolved in one case with a complex pathogenic rearrangement affecting TSC2. Conclusion Our findings demonstrate that long-read sequencing can improve the validation, resolution, and classification of germline SVs. This has important implications for return of results, cascade carrier testing, cancer screening, and prophylactic interventions.
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Craddock, Hillary A., Yair Motro, Bar Zilberman, Boris Khalfin, Svetlana Bardenstein, and Jacob Moran-Gilad. "Long-Read Sequencing and Hybrid Assembly for Genomic Analysis of Clinical Brucella melitensis Isolates." Microorganisms 10, no. 3 (2022): 619. http://dx.doi.org/10.3390/microorganisms10030619.
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Brucella melitensis is a key etiological agent of brucellosis and has been increasingly subject to characterization using sequencing methodologies. This study aimed to investigate and compare short-read, long-read, and hybrid assemblies of B. melitensis. Eighteen B. melitensis isolates from Southern Israel were sequenced using Illumina and the Oxford Nanopore (ONP) MinION, and hybrid assemblies were generated with ONP long reads scaffolded on Illumina short reads. Short reads were assembled with INNUca with SPADes, long reads and hybrid with dragonflye. Abricate with the virulence factor database (VFDB) and in silico PCR (for the genes BetB, BPE275, BSPB, manA, mviN, omp19, perA, PrpA, VceC, and ureI) were used for identifying virulence genes, and a total of 61 virulence genes were identified in short-read, long-read, and hybrid assemblies of all 18 isolates. The phylogenetic analysis using long-read assemblies revealed several inconsistencies in cluster assignment as compared to using hybrid and short-read assemblies. Overall, hybrid assembly provided the most comprehensive data, and stand-alone short-read sequencing provided comparable data to stand-alone long-read sequencing regarding virulence genes. For genomic epidemiology studies, stand-alone ONP sequencing may require further refinement in order to be useful in endemic settings.
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Kainth, Amoldeep S., Gabriela A. Haddad, Johnathon M. Hall, and Alexander J. Ruthenburg. "Merging short and stranded long reads improves transcript assembly." PLOS Computational Biology 19, no. 10 (2023): e1011576. http://dx.doi.org/10.1371/journal.pcbi.1011576.
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Long-read RNA sequencing has arisen as a counterpart to short-read sequencing, with the potential to capture full-length isoforms, albeit at the cost of lower depth. Yet this potential is not fully realized due to inherent limitations of current long-read assembly methods and underdeveloped approaches to integrate short-read data. Here, we critically compare the existing methods and develop a new integrative approach to characterize a particularly challenging pool of low-abundance long noncoding RNA (lncRNA) transcripts from short- and long-read sequencing in two distinct cell lines. Our analysis reveals severe limitations in each of the sequencing platforms. For short-read assemblies, coverage declines at transcript termini resulting in ambiguous ends, and uneven low coverage results in segmentation of a single transcript into multiple transcripts. Conversely, long-read sequencing libraries lack depth and strand-of-origin information in cDNA-based methods, culminating in erroneous assembly and quantitation of transcripts. We also discover a cDNA synthesis artifact in long-read datasets that markedly impacts the identity and quantitation of assembled transcripts. Towards remediating these problems, we develop a computational pipeline to “strand” long-read cDNA libraries that rectifies inaccurate mapping and assembly of long-read transcripts. Leveraging the strengths of each platform and our computational stranding, we also present and benchmark a hybrid assembly approach that drastically increases the sensitivity and accuracy of full-length transcript assembly on the correct strand and improves detection of biological features of the transcriptome. When applied to a challenging set of under-annotated and cell-type variable lncRNA, our method resolves the segmentation problem of short-read sequencing and the depth problem of long-read sequencing, resulting in the assembly of coherent transcripts with precise 5’ and 3’ ends. Our workflow can be applied to existing datasets for superior demarcation of transcript ends and refined isoform structure, which can enable better differential gene expression analyses and molecular manipulations of transcripts.
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Volden, Roger, Theron Palmer, Ashley Byrne, et al. "Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA." Proceedings of the National Academy of Sciences 115, no. 39 (2018): 9726–31. http://dx.doi.org/10.1073/pnas.1806447115.
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High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. While long-read sequencing technology can provide this long-distance information, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here, we introduce and validate a long-read ONT-based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single-cell samples.
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Limberis, Jason D., Roland J. Nagel, Soumitesh Chakravorty, et al. "stilPCR increases the effective sequencing length of Illumina targeted next-generation sequencing." PLOS ONE 19, no. 12 (2024): e0314265. https://doi.org/10.1371/journal.pone.0314265.
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Identifying pathogens, resistance-conferring mutations, and strain types through targeted amplicon sequencing is an important tool. However, due to the limitations of short read sequencing, many applications require the division of limited clinical samples. Here, we present stilPCR (single-tube Illumina long read PCR), which allows the generation of hemi-nested amplicons in a single tube, with Illumina indexes and adapters, effectively increasing the Illumina read length without increasing the input requirements of reagents or sample. We have successfully utilized stilPCR on clinical sputum from tuberculosis patients to detect drug resistance mutations.
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Greenman, Noah, Sayf Al-Deen Hassouneh, Latifa S. Abdelli, Catherine Johnston, and Taj Azarian. "Improving Bacterial Metagenomic Research through Long-Read Sequencing." Microorganisms 12, no. 5 (2024): 935. http://dx.doi.org/10.3390/microorganisms12050935.
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Metagenomic sequencing analysis is central to investigating microbial communities in clinical and environmental studies. Short-read sequencing remains the primary approach for metagenomic research; however, long-read sequencing may offer advantages of improved metagenomic assembly and resolved taxonomic identification. To compare the relative performance for metagenomic studies, we simulated short- and long-read datasets using increasingly complex metagenomes comprising 10, 20, and 50 microbial taxa. Additionally, we used an empirical dataset of paired short- and long-read data generated from mouse fecal pellets to assess real-world performance. We compared metagenomic assembly quality, taxonomic classification, and metagenome-assembled genome (MAG) recovery rates. We show that long-read sequencing data significantly improve taxonomic classification and assembly quality. Metagenomic assemblies using simulated long reads were more complete and more contiguous with higher rates of MAG recovery. This resulted in more precise taxonomic classifications. Principal component analysis of empirical data demonstrated that sequencing technology affects compositional results as samples clustered by sequence type, not sample type. Overall, we highlight strengths of long-read metagenomic sequencing for microbiome studies, including improving the accuracy of classification and relative abundance estimates. These results will aid researchers when considering which sequencing approaches to use for metagenomic projects.
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Iyer, Shruti V., Sara Goodwin, and William Richard McCombie. "Leveraging the power of long reads for targeted sequencing." Genome Research 34, no. 11 (2024): 1701–18. http://dx.doi.org/10.1101/gr.279168.124.
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Long-read sequencing technologies have improved the contiguity and, as a result, the quality of genome assemblies by generating reads long enough to span and resolve complex or repetitive regions of the genome. Several groups have shown the power of long reads in detecting thousands of genomic and epigenomic features that were previously missed by short-read sequencing approaches. While these studies demonstrate how long reads can help resolve repetitive and complex regions of the genome, they also highlight the throughput and coverage requirements needed to accurately resolve variant alleles across large populations using these platforms. At the time of this review, whole-genome long-read sequencing is more expensive than short-read sequencing on the highest throughput short-read instruments; thus, achieving sufficient coverage to detect low-frequency variants (such as somatic variation) in heterogenous samples remains challenging. Targeted sequencing, on the other hand, provides the depth necessary to detect these low-frequency variants in heterogeneous populations. Here, we review currently used and recently developed targeted sequencing strategies that leverage existing long-read technologies to increase the resolution with which we can look at nucleic acids in a variety of biological contexts.
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Gilmore, Barbara S., Nahla V. Bassil, Danny L. Barney, Brian J. Knaus, and Kim E. Hummer. "Short-read DNA Sequencing Yields Microsatellite Markers for Rheum." Journal of the American Society for Horticultural Science 139, no. 1 (2014): 22–29. http://dx.doi.org/10.21273/jashs.139.1.22.
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Identifying and evaluating genetic diversity of culinary rhubarb (Rheum ×rhababarum) cultivars using morphological characteristics is challenging given the existence of synonyms and nomenclatural inconsistencies. Some cultivars with similar names are morphologically different, and seedlings may grow and become associated with the parental name. Morphological traits of one cultivar may vary when measured under different environmental conditions. Molecular markers are consistent for unique genotypes across environments and provide genetic fingerprints to assist in resolving identity issues. Microsatellite repeats, also called simple sequence repeats (SSRs), are commonly used for fingerprinting fruit and nut crops, but only 10 SSRs have previously been reported in rhubarb. The objectives of this study were to use short-read DNA sequences to develop new di-nucleotide-containing SSR markers for rhubarb and to determine if the markers were useful for cultivar identification. A total of 97 new SSR primer pairs were designed from the short-read DNA sequences. The amplification success rate of these SSRs was 77%, whereas polymorphism of those reached 76% in a test panel of four or eight rhubarb individuals. From the 57 potentially polymorphic primer pairs obtained, 25 SSRs were evaluated in 58 Rheum accessions preserved in the U.S. Department of Agriculture, National Plant Germplasm System. The primer pairs generated 314 fragments with an average of 12.6 fragments per pair. The clustering of many accessions in well-supported groups supported previous findings based on amplified fragment length polymorphisms (AFLPs). Cluster analysis, using the proportion of shared allele distance among the 25 SSRs, distinguished each of the 58 accessions including individuals that had similar names or the same name. Accessions that grouped in well-supported clusters previously belonged to similar clusters with high bootstrap support based on AFLP. In summary, our technique of mining short-read sequencing data was successful in identifying 97 di-nucleotide-containing SSR sequences. Of those tested, the 25 most polymorphic and easy-to-score primer pairs proved useful in fingerprinting rhubarb cultivars. We recommend the use of short-read sequencing for the development of SSR markers in the identification of horticultural crops.
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Solomon, Brad, and Carl Kingsford. "Fast search of thousands of short-read sequencing experiments." Nature Biotechnology 34, no. 3 (2016): 300–302. http://dx.doi.org/10.1038/nbt.3442.
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Stapleton, James A., Jeongwoon Kim, John P. Hamilton, et al. "Haplotype-Phased Synthetic Long Reads from Short-Read Sequencing." PLOS ONE 11, no. 1 (2016): e0147229. http://dx.doi.org/10.1371/journal.pone.0147229.
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Whiteford, N. "An analysis of the feasibility of short read sequencing." Nucleic Acids Research 33, no. 19 (2005): e171-e171. http://dx.doi.org/10.1093/nar/gni170.
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Wick, Ryan R., Louise M. Judd, and Kathryn E. Holt. "Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing." PLOS Computational Biology 19, no. 3 (2023): e1010905. http://dx.doi.org/10.1371/journal.pcbi.1010905.
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A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism’s genome—each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers, and polishers have brought perfect assemblies within reach. Here, we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools and manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).
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Wei, Po-Li, Ching-Sheng Hung, Yi-Wei Kao, et al. "Characterization of Fecal Microbiota with Clinical Specimen Using Long-Read and Short-Read Sequencing Platform." International Journal of Molecular Sciences 21, no. 19 (2020): 7110. http://dx.doi.org/10.3390/ijms21197110.
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Accurate and rapid identification of microbiotic communities using 16S ribosomal (r)RNA sequencing is a critical task for expanding medical and clinical applications. Next-generation sequencing (NGS) is widely considered a practical approach for direct application to communities without the need for in vitro culturing. In this report, a comparative evaluation of short-read (Illumina) and long-read (Oxford Nanopore Technologies (ONT)) platforms toward 16S rRNA sequencing with the same batch of total genomic DNA extracted from fecal samples is presented. Different 16S gene regions were amplified, bar-coded, and sequenced using the Illumina MiSeq and ONT MinION sequencers and corresponding kits. Mapping of the sequenced amplicon using MinION to the entire 16S rRNA gene was analyzed with the cloud-based EPI2ME algorithm. V3–V4 reads generated using MiSeq were aligned by applying the CLC genomics workbench. More than 90% of sequenced reads generated using distinct sequencers were accurately classified at the genus or species level. The misclassification of sequenced reads at the species level between the two approaches was less substantial as expected. Taken together, the comparative results demonstrate that MinION sequencing platform coupled with the corresponding algorithm could function as a practicable strategy in classifying bacterial community to the species level.
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Santos, Renato, Hyunah Lee, Alexander Williams, et al. "Investigating the Performance of Oxford Nanopore Long-Read Sequencing with Respect to Illumina Microarrays and Short-Read Sequencing." International Journal of Molecular Sciences 26, no. 10 (2025): 4492. https://doi.org/10.3390/ijms26104492.
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Oxford Nanopore Technologies (ONT) long-read sequencing (LRS) has emerged as a promising genomic analysis tool, yet comprehensive benchmarks with established platforms across diverse datasets remain limited. This study aimed to benchmark LRS performance against Illumina short-read sequencing (SRS) and microarrays for variant detection across different genomic contexts and to evaluate the impact of experimental factors. We sequenced 14 human genomes using the three platforms and evaluated single nucleotide variants (SNVs), insertions/deletions (indels), and structural variants (SVs) detection, stratifying by high-complexity, low-complexity, and dark genome regions while assessing effects of multiplexing, depth, and read length. LRS SNV accuracy was slightly lower than that of SRS in high-complexity regions (F-measure: 0.954 vs. 0.967) but showed comparable sensitivity in low-complexity regions. LRS showed robust performance for small (1–5 bp) indels in high-complexity regions (F-measure: 0.869), but SRS agreement decreased significantly in low-complexity regions and for larger indel sizes. Within dark regions, LRS identified more indels than SRS, but showed lower base-level accuracy. LRS identified 2.86 times more SVs than SRS, excelling at detecting large variants (>6 kb), with SV detection improving with sequencing depth. Sequencing depth strongly influenced variant calling performance, whereas multiplexing effects were minimal. Our findings provide valuable insights for optimising LRS applications in genomic research and diagnostics.
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Kaplun, Ludmila, Greice Krautz-Peterson, Nir Neerman, et al. "ONT in Clinical Diagnostics of Repeat Expansion Disorders: Detection and Reporting Challenges." International Journal of Molecular Sciences 26, no. 6 (2025): 2725. https://doi.org/10.3390/ijms26062725.
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While whole-genome sequencing (WGS) using short-read technology has become a standard diagnostic test, this technology has limitations in analyzing certain genomic regions, particularly short tandem repeats (STRs). These repetitive sequences are associated with over 50 diseases, primarily affecting neurological function, including Huntington disease, frontotemporal dementia, and Friedreich’s ataxia. We analyzed 2689 cases with movement disorders and dementia-related phenotypes processed at Variantyx in 2023–2024 using a two-tiered approach, with an initial short-read WGS followed by ONT long-read sequencing (when necessary) for variant characterization. Of the 2038 cases (75.8%) with clinically relevant genetic variants, 327 (16.0%) required additional long-read analysis. STR variants were reported in 338 cases (16.6% of positive cases), with approximately half requiring long-read sequencing for definitive classification. The combined approach enabled the precise determination of repeat length, composition, somatic mosaicism, and methylation status. Notable advantages included the detection of complex repeat structures in several genes such as RFC1, FGF14, and FXN, where long-read sequencing allowed to determine somatic repeat unit variations and accurate allele phasing. Further studies are needed to establish technology-specific guidelines for the standardized interpretation of long-read sequencing data for the clinical diagnostics of repeat expansion disorders.
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Han, Seungbin, Nazia Afrin, Bas Tolhuis, et al. "Implementing Hifi Sequencing for Enhanced Genomic and Methylome Characterization in Multiple Myeloma." Blood 144, Supplement 1 (2024): 7490. https://doi.org/10.1182/blood-2024-207422.
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Introduction: Over recent years, the introduction and scalability of next-generation sequencing technologies have significantly expanded our knowledge of the genetic underpinnings of multiple myeloma (MM), a heterogeneous hematologic malignancy characterized by clonal plasma cell proliferation in the bone marrow. At present, cost-effective short-read technologies are extensively utilized for sequencing. Nevertheless, these platforms are limited in their ability to fully capture repeated regions and structural variants. Progress in long-read sequencing technologies, including HiFi-sequencing (HiFi), produces extended reads with increased precision, enabling more comprehensive and continuous genome assemblies. Another notable benefit of HiFi is its ability to integrate genome and methylome data, which seems increasingly needed given that epigenetic regulation has been identified as a major driver of resistance in MM. Methods: We collected CD138+ cells from the bone marrow of four newly diagnosed and nine relapsed / refractory MM patients. Sequencing of all samples was performed with a coverage of 30x using HiFi sequencing. In addition, we conducted short-read sequencing with 100x coverage to facilitate a direct comparison between the two approaches. We performed in-depth coverage analysis using mosdepth to compare the read mappability of the two sequencing technologies. Comparative downstream analysis, with a specific focus on structural variations (SVs), single nucleotide variations (SNVs), and methylome analysis was performed. Results: The goal of this study was to conduct a technical feasibility analysis to examine genome coverage as well as detection of structural variants (SVs) and single nucleotide variants (SNVs) by HiFi long-read sequencing and short-read sequencing in a cohort of MM patients with heterogeneous disease activity. As anticipated, a mean genome coverage of 30.1x was achieved by HiFi-sequencing across all samples. Read length was consistently >10,000 bp with a mean of 13,047 bp (range: 10,503-14,836). The median quality value (QV) for each base in the sequence was reassuring, with values ranging between 34 and 40. An average of 5,658,975 regions with high coverage (each region = 500 bp) were seen by HiFi sequencing as compared to 5,620,710 regions by short-read sequencing , resulting in a difference of 19.1 Mbp with improved coverage. By applying ClairS-TO, a deep-learning algorithm to uncover somatic mutations, we could observe that the HiFi technology could detect more somatic variants (n = 151,555) as compared to the short-read approach (n = 129,921). Structural variant calling and analysis are currently ongoing and will be performed by established tools for long-read vs. short-read analysis. Furthermore, we acquired healthy plasma cells from 13 individuals and sequenced with HiFiseq. The metyhlome and genome of these samples will serve as the reference for further comparison analysis with multiple myeloma patient samples through which we will provide insights into the MM cells specific genome-wide methylattion profile as well as genomics events. Summary: In summary, we here report on the feasibility of HiFi sequencing for enhanced genomic and methylome characterization in MM. Our final analysis is ongoing and updated results will be presented at the meeting.
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Wong, Kwong-Kwok, Yvonne Tsang, and David M. Gershenson. "Abstract 4081: Analysis of low-grade serous ovarian cancer by long-read full length transcripts sequencing." Cancer Research 85, no. 8_Supplement_1 (2025): 4081. https://doi.org/10.1158/1538-7445.am2025-4081.
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Abstract Low grade serous ovarian carcinoma is a rare epithelial ovarian cancer that occurs more frequently in younger women. RNA sequencing (RNAseq) has been playing a pivotal role in understanding the molecular pathogenesis of the low-grade ovarian serous carcinoma (LGSOC). The quantification of gene expression with enough sequencing depth can be fairly accurate. However, the current short-read RNAseq approach is not very accurate in measuring individual transcript activity. This is because multiple transcripts from the same gene share high sequence similarity, which complicates the transcript mapping. As the accuracy of long-read sequencing technology is improving, long-read full transcript RNA sequencing is becoming an important tool for interrogating alternative transcripts and the discovery of gene fusion transcripts. In this study, we compare short-read RNAseq and long-read RNAseq from the same LGSOC patient samples. The RNA-Seq analysis for long-reads were performed by mapping sequencing reads to annotated reference human genome GrCh38 with minimap2. First, all annotated transcripts or genes were extracted including any annotated splice variants. Subsequently, the reads were mapped against all the transcripts as well as to the whole genome using minimap2. The distributing and counting the reads across genes and transcripts were calculated. We found that the expression level of transcripts in short-read in general is higher than that that of long-read. It is because short reads frequently could not be accurately mapped to the correct alternative transcripts from the same genes. When a read mapped to multiple transcripts, the short reads were randomly assigned to a transcript from the same gene. As a result, more than 100, 000 transcripts had at least one assigned read in the short-read RNAseq analysis, but less than 50, 000 transcripts had at least one assigned read in long-read RNAseq analysis. Long read with greater read-depth had better quantification accuracy for detecting specific transcripts. To identify potential gene fusions, the raw Oxford Nanopore long-read RNA-seq fastq files were corrected and polished with high sequencing quality short-read RNA-seq data by racon assembly tool. Those long-reads with unaligned ends of reads from a mapping generated by RNA-Seq analysis were re-mapped to the reference using the unaligned ends of reads. In this study, we identified several gene fusions including a gene fusion between NSUN4 and FAAH genes with at least seven long reads covering the breakpoint. FAAH encodes the enzyme fatty acid amide hydrolase and has been shown to be a metastasis suppressor in breast cancer previously. It is possible that the truncated FAAH protein because of gene fusion is involved in the metastasis of the LGSOC that we have analyzed. This could be another mechanism involved in the progression of LGSOC. Additional analysis of the long-read full transcript sequencing of LGSOC is in progress. Citation Format: Kwong-Kwok Wong, Yvonne Tsang, David M. Gershenson. Analysis of low-grade serous ovarian cancer by long-read full length transcripts sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4081.
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Caspar, Sylvan Manuel, Timo Schneider, Patricia Stoll, Janine Meienberg, and Gabor Matyas. "Potential of whole-genome sequencing-based pharmacogenetic profiling." Pharmacogenomics 22, no. 3 (2021): 177–90. http://dx.doi.org/10.2217/pgs-2020-0155.
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Pharmacogenetics represents a major driver of precision medicine, promising individualized drug selection and dosing. Traditionally, pharmacogenetic profiling has been performed using targeted genotyping that focuses on common/known variants. Recently, whole-genome sequencing (WGS) is emerging as a more comprehensive short-read next-generation sequencing approach, enabling both gene diagnostics and pharmacogenetic profiling, including rare/novel variants, in a single assay. Using the example of the pharmacogene CYP2D6, we demonstrate the potential of WGS-based pharmacogenetic profiling as well as emphasize the limitations of short-read next-generation sequencing. In the near future, we envision a shift toward long-read sequencing as the predominant method for gene diagnostics and pharmacogenetic profiling, providing unprecedented data quality and improving patient care.
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Sereika, Mantas, Rasmus Hansen Kirkegaard, Søren Michael Karst, et al. "Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing." Nature Methods 19, no. 7 (2022): 823–26. http://dx.doi.org/10.1038/s41592-022-01539-7.
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AbstractLong-read Oxford Nanopore sequencing has democratized microbial genome sequencing and enables the recovery of highly contiguous microbial genomes from isolates or metagenomes. However, to obtain near-finished genomes it has been necessary to include short-read polishing to correct insertions and deletions derived from homopolymer regions. Here, we show that Oxford Nanopore R10.4 can be used to generate near-finished microbial genomes from isolates or metagenomes without short-read or reference polishing.
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Mirus, Tim, Robert Lohmayer, Clementine Döhring, Bjarni V. Halldórsson, and Birte Kehr. "GGTyper: genotyping complex structural variants using short-read sequencing data." Bioinformatics 40, Supplement_2 (2024): ii11—ii19. http://dx.doi.org/10.1093/bioinformatics/btae391.
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Abstract Motivation Complex structural variants (SVs) are genomic rearrangements that involve multiple segments of DNA. They contribute to human diversity and have been shown to cause Mendelian disease. Nevertheless, our abilities to analyse complex SVs are very limited. As opposed to deletions and other canonical types of SVs, there are no established tools that have explicitly been designed for analysing complex SVs. Results Here, we describe a new computational approach that we specifically designed for genotyping complex SVs in short-read sequenced genomes. Given a variant description, our approach computes genotype-specific probability distributions for observing aligned read pairs with a wide range of properties. Subsequently, these distributions can be used to efficiently determine the most likely genotype for any set of aligned read pairs observed in a sequenced genome. In addition, we use these distributions to compute a genotyping difficulty for a given variant, which predicts the amount of data needed to achieve a reliable call. Careful evaluation confirms that our approach outperforms other genotypers by making reliable genotype predictions across both simulated and real data. On up to 7829 human genomes, we achieve high concordance with population-genetic assumptions and expected inheritance patterns. On simulated data, we show that precision correlates well with our prediction of genotyping difficulty. This together with low memory and time requirements makes our approach well-suited for application in biomedical studies involving small to very large numbers of short-read sequenced genomes. Availability and implementation Source code is available at https://github.com/kehrlab/Complex-SV-Genotyping.
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Zhang, Pengfei, Dike Jiang, Yin Wang, Xueping Yao, Yan Luo, and Zexiao Yang. "Comparison of De Novo Assembly Strategies for Bacterial Genomes." International Journal of Molecular Sciences 22, no. 14 (2021): 7668. http://dx.doi.org/10.3390/ijms22147668.
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(1) Background: Short-read sequencing allows for the rapid and accurate analysis of the whole bacterial genome but does not usually enable complete genome assembly. Long-read sequencing greatly assists with the resolution of complex bacterial genomes, particularly when combined with short-read Illumina data. However, it is not clear how different assembly strategies affect genomic accuracy, completeness, and protein prediction. (2) Methods: we compare different assembly strategies for Haemophilus parasuis, which causes Glässer’s disease, characterized by fibrinous polyserositis and arthritis, in swine by using Illumina sequencing and long reads from the sequencing platforms of either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio). (3) Results: Assembly with either PacBio or ONT reads, followed by polishing with Illumina reads, facilitated high-quality genome reconstruction and was superior to the long-read-only assembly and hybrid-assembly strategies when evaluated in terms of accuracy and completeness. An equally excellent method was correction with Homopolish after the ONT-only assembly, which had the advantage of avoiding hybrid sequencing with Illumina. Furthermore, by aligning transcripts to assembled genomes and their predicted CDSs, the sequencing errors of the ONT assembly were mainly indels that were generated when homopolymer regions were sequenced, thus critically affecting protein prediction. Polishing can fill indels and correct mistakes. (4) Conclusions: The assembly of bacterial genomes can be directly achieved by using long-read sequencing techniques. To maximize assembly accuracy, it is essential to polish the assembly with homologous sequences of related genomes or sequencing data from short-read technology.
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Shumate, Alaina, Brandon Wong, Geo Pertea, and Mihaela Pertea. "Improved transcriptome assembly using a hybrid of long and short reads with StringTie." PLOS Computational Biology 18, no. 6 (2022): e1009730. http://dx.doi.org/10.1371/journal.pcbi.1009730.
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Short-read RNA sequencing and long-read RNA sequencing each have their strengths and weaknesses for transcriptome assembly. While short reads are highly accurate, they are rarely able to span multiple exons. Long-read technology can capture full-length transcripts, but its relatively high error rate often leads to mis-identified splice sites. Here we present a new release of StringTie that performs hybrid-read assembly. By taking advantage of the strengths of both long and short reads, hybrid-read assembly with StringTie is more accurate than long-read only or short-read only assembly, and on some datasets it can more than double the number of correctly assembled transcripts, while obtaining substantially higher precision than the long-read data assembly alone. Here we demonstrate the improved accuracy on simulated data and real data from Arabidopsis thaliana, Mus musculus, and human. We also show that hybrid-read assembly is more accurate than correcting long reads prior to assembly while also being substantially faster. StringTie is freely available as open source software at https://github.com/gpertea/stringtie.
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Sockell, Alexandra, Khi Pin Chua, Christopher Kingsley, et al. "Abstract 6624: Comprehensive, multi-omic detection of somatic variants from the GIAB HG008 matched tumor-normal pair using highly accurate long- and short-read whole-genome sequencing." Cancer Research 85, no. 8_Supplement_1 (2025): 6624. https://doi.org/10.1158/1538-7445.am2025-6624.
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Abstract Disentangling the molecular drivers of cancer progression requires a precise understanding of the somatic alterations that take place at the DNA level during tumor development. These include not only small changes like SNVs and indels, but also structural variants, changes in repetitive elements, differential methylation, as well as the haplotype context in which these changes occur. Existing short-read sequencing methods using sequencing by synthesis (SBS) chemistry lack the read length to characterize large structural variants or to span longer repetitive regions as well as to phase variants into haplotypes, while simultaneously lacking the sequencing accuracy to distinguish very low frequency somatic variants from background sequencing error or to sequence through shorter repetitive elements like microsatellites. Long-read PacBio HiFi data addresses the first set of challenges by accurately sequencing long fragments up to 25 kb, while highly accurate short-read sequencing by binding (SBB) chemistry from PacBio addresses the latter by improving raw sequencing accuracy by orders of magnitude, including in homopolymer regions which make up most microsatellites in the human genome. Here we apply these highly accurate long- and short-read sequencing technologies from PacBio to perform whole-genome sequencing of the newly described HG008 matched tumor-normal pair from the Genome in a Bottle (GIAB) consortium. This reference sample includes an adherent, epithelial-like pancreatic adenocarcinoma (PDAC) cell line as the tumor material, with the matched normal obtained from adjacent duodenal and pancreatic tissue. We perform whole-genome sequencing with both PacBio HiFi (35X normal, 80X tumor) and SBB (50X normal, 100X tumor), followed by alignment and tumor-normal variant calling for all variant types. These accurate long- and short-read sequencing technologies offer a more robust and comprehensive picture of somatic variation in this reference sample and contribute to developing this novel benchmark. Citation Format: Alexandra Sockell, Khi Pin Chua, Christopher Kingsley, Dan Nasko, Matthew Boitano, Young Kim, Melanie Wescott, Ian McLaughlin, Primo Baybayan, Jennifer McDaniel, Andrew Shaver, Justin M. Zook, Aaron Wenger. Comprehensive, multi-omic detection of somatic variants from the GIAB HG008 matched tumor-normal pair using highly accurate long- and short-read whole-genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6624.
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Kumar, Kishore R., Mark J. Cowley, and Ryan L. Davis. "Next-Generation Sequencing and Emerging Technologies." Seminars in Thrombosis and Hemostasis 45, no. 07 (2019): 661–73. http://dx.doi.org/10.1055/s-0039-1688446.
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AbstractGenetic sequencing technologies are evolving at a rapid pace with major implications for research and clinical practice. In this review, the authors provide an updated overview of next-generation sequencing (NGS) and emerging methodologies. NGS has tremendously improved sequencing output while being more time and cost-efficient in comparison to Sanger sequencing. The authors describe short-read sequencing approaches, such as sequencing by synthesis, ion semiconductor sequencing, and nanoball sequencing. Third-generation long-read sequencing now promises to overcome many of the limitations of short-read sequencing, such as the ability to reliably resolve repeat sequences and large genomic rearrangements. By combining complementary methods with massively parallel DNA sequencing, a greater insight into the biological context of disease mechanisms is now possible. Emerging methodologies, such as advances in nanopore technology, in situ nucleic acid sequencing, and microscopy-based sequencing, will continue the rapid evolution of this area. These new technologies hold many potential applications for hematological disorders, with the promise of precision and personalized medical care in the future.
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Begum, Ghausia, Ammar Albanna, Asma Bankapur, et al. "Long-Read Sequencing Improves the Detection of Structural Variations Impacting Complex Non-Coding Elements of the Genome." International Journal of Molecular Sciences 22, no. 4 (2021): 2060. http://dx.doi.org/10.3390/ijms22042060.
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The advent of long-read sequencing offers a new assessment method of detecting genomic structural variation (SV) in numerous rare genetic diseases. For autism spectrum disorders (ASD) cases where pathogenic variants fail to be found in the protein-coding genic regions along chromosomes, we proposed a scalable workflow to characterize the risk factor of SVs impacting non-coding elements of the genome. We applied whole-genome sequencing on an Emirati family having three children with ASD using long and short-read sequencing technology. A series of analytical pipelines were established to identify a set of SVs with high sensitivity and specificity. At 15-fold coverage, we observed that long-read sequencing technology (987 variants) detected a significantly higher number of SVs when compared to variants detected using short-read technology (509 variants) (p-value < 1.1020 × 10−57). Further comparison showed 97.9% of long-read sequencing variants were spanning within the 1–100 kb size range (p-value < 9.080 × 10−67) and impacting over 5000 genes. Moreover, long-read variants detected 604 non-coding RNAs (p-value < 9.02 × 10−9), comprising 58% microRNA, 31.9% lncRNA, and 9.1% snoRNA. Even at low coverage, long-read sequencing has shown to be a reliable technology in detecting SVs impacting complex elements of the genome.
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Gouil, Quentin, and Andrew Keniry. "Latest techniques to study DNA methylation." Essays in Biochemistry 63, no. 6 (2019): 639–48. http://dx.doi.org/10.1042/ebc20190027.
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Abstract Bisulfite sequencing is a powerful technique to detect 5-methylcytosine in DNA that has immensely contributed to our understanding of epigenetic regulation in plants and animals. Meanwhile, research on other base modifications, including 6-methyladenine and 4-methylcytosine that are frequent in prokaryotes, has been impeded by the lack of a comparable technique. Bisulfite sequencing also suffers from a number of drawbacks that are difficult to surmount, among which DNA degradation, lack of specificity, or short reads with low sequence diversity. In this review, we explore the recent refinements to bisulfite sequencing protocols that enable targeting genomic regions of interest, detecting derivatives of 5-methylcytosine, and mapping single-cell methylomes. We then present the unique advantage of long-read sequencing in detecting base modifications in native DNA and highlight the respective strengths and weaknesses of PacBio and Nanopore sequencing for this application. Although analysing epigenetic data from long-read platforms remains challenging, the ability to detect various modified bases from a universal sample preparation, in addition to the mapping and phasing advantages of the longer read lengths, provide long-read sequencing with a decisive edge over short-read bisulfite sequencing for an expanding number of applications across kingdoms.
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Chen, Nancy, Daniel W. Bellott, David C. Page, and Andrew G. Clark. "Identification of avian W-linked contigs by short-read sequencing." BMC Genomics 13, no. 1 (2012): 183. http://dx.doi.org/10.1186/1471-2164-13-183.
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Lefrançois, Philippe, Ghia M. Euskirchen, Raymond K. Auerbach, et al. "Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing." BMC Genomics 10, no. 1 (2009): 37. http://dx.doi.org/10.1186/1471-2164-10-37.
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Fang, Han, Ewa A. Bergmann, Kanika Arora, et al. "Indel variant analysis of short-read sequencing data with Scalpel." Nature Protocols 11, no. 12 (2016): 2529–48. http://dx.doi.org/10.1038/nprot.2016.150.
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Hunter, Ben, Kevin M. Wright, and Kirsten Bomblies. "Short read sequencing in studies of natural variation and adaptation." Current Opinion in Plant Biology 16, no. 1 (2013): 85–91. http://dx.doi.org/10.1016/j.pbi.2012.10.003.
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Chu, Hsueh-Ting, William WL Hsiao, Theresa TH Tsao, et al. "SeqEntropy: Genome-Wide Assessment of Repeats for Short Read Sequencing." PLoS ONE 8, no. 3 (2013): e59484. http://dx.doi.org/10.1371/journal.pone.0059484.
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Chen, Xinyue, Xiaodong Lu, Xianglin Sh, Shaojun Yu, and Jonathan Zhao. "Long-Read Sequencing Outperforms Short-Read Sequencing in Detecting Most Structural Variations." Serican Journal of Medicine 2, no. 2 (2025). https://doi.org/10.17161/sjm.v2i2.23671.
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Structural variations (SV) are common in the cancer genome and play critical roles in regulating tumorigenesis. In the past decades, many SVs have been detected through analyses of whole-genome sequencing (WGS) data generated mainly by Illumina paired-end short-read sequencing (SRS). Recent advances in long-read sequencing (LRS) techniques provide exciting opportunities for SV detection. However, a comprehensive analysis of the pros and cons of LRS and SRS in detecting SVs in a cancer genome is still lacking. Here, we performed WGS of the LNCaP prostate cancer cell line through LRS using the Oxford Nanopore Technology and called main SVs, which were compared to those derived from publicly available LNCaP SRS data. Strikingly, LRS is superior in detecting insertions of all sizes and deletions of <1000 bp long, whereas SRS is very useful in capturing long deletions, taking advantage of its paired-end reads. LRS identified more precise breakpoints of detected SVs. In addition, we found that SRS called many duplications and inversions, most of which were not confirmed by LRS, likely due to ambiguity in SRS read alignment to repetitive regions, leading to errors in SV calling. In conclusion, LRS outperformed SRS in detecting most SVs, except deletions longer than LRS read lengths. Our study highlights the advantages of LRS in resolving complex genomic rearrangements and underscores its potential for improving SV detection in cancer genomics.
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Adewale, Boluwatife A. "Will long-read sequencing technologies replace short-read sequencing technologies in the next 10 years?" African Journal of Laboratory Medicine 9, no. 1 (2020). http://dx.doi.org/10.4102/ajlm.v9i1.1340.
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Orellana, Luis H., Karen Krüger, Chandni Sidhu, and Rudolf Amann. "Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies." Microbiome 11, no. 1 (2023). http://dx.doi.org/10.1186/s40168-023-01557-3.
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Abstract Background Over the past years, sequencing technologies have expanded our ability to examine novel microbial metabolisms and diversity previously obscured by isolation approaches. Long-read sequencing promises to revolutionize the metagenomic field and recover less fragmented genomes from environmental samples. Nonetheless, how to best benefit from long-read sequencing and whether long-read sequencing can provide recovered genomes of similar characteristics as short-read approaches remains unclear. Results We recovered metagenome-assembled genomes (MAGs) from the free-living fraction at four-time points during a spring bloom in the North Sea. The taxonomic composition of all MAGs recovered was comparable between technologies. However, differences consisted of higher sequencing depth for contigs and higher genome population diversity in short-read compared to long-read metagenomes. When pairing population genomes recovered from both sequencing approaches that shared ≥ 99% average nucleotide identity, long-read MAGs were composed of fewer contigs, a higher N50, and a higher number of predicted genes when compared to short-read MAGs. Moreover, 88% of the total long-read MAGs carried a 16S rRNA gene compared to only 23% of MAGs recovered from short-read metagenomes. Relative abundances for population genomes recovered using both technologies were similar, although disagreements were observed for high and low GC content MAGs. Conclusions Our results highlight that short-read technologies recovered more MAGs and a higher number of species than long-read due to an overall higher sequencing depth. Long-read samples produced higher quality MAGs and similar species composition compared to short-read sequencing. Differences in the GC content recovered by each sequencing technology resulted in divergences in the diversity recovered and relative abundance of MAGs within the GC content boundaries.
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Liu, Silvia, Caroline Obert, Yan-Ping Yu, et al. "Utility analyses of AVITI sequencing chemistry." BMC Genomics 25, no. 1 (2024). http://dx.doi.org/10.1186/s12864-024-10686-4.
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Abstract Background DNA sequencing is a critical tool in modern biology. Over the last two decades, it has been revolutionized by the advent of massively parallel sequencing, leading to significant advances in the genome and transcriptome sequencing of various organisms. Nevertheless, challenges with accuracy, lack of competitive options and prohibitive costs associated with high throughput parallel short-read sequencing persist. Results Here, we conduct a comparative analysis using matched DNA and RNA short-reads assays between Element Biosciences’ AVITI and Illumina’s NextSeq 550 chemistries. Similar comparisons were evaluated for synthetic long-read sequencing for RNA and targeted single-cell transcripts between the AVITI and Illumina’s NovaSeq 6000. For both DNA and RNA short-read applications, the study found that the AVITI produced significantly higher per sequence quality scores. For PCR-free DNA libraries, we observed an average 89.7% lower experimentally determined error rate when using the AVITI chemistry, compared to the NextSeq 550. For short-read RNA quantification, AVITI platform had an average of 32.5% lower error rate than that for NextSeq 550. With regards to synthetic long-read mRNA and targeted synthetic long read single cell mRNA sequencing, both platforms’ respective chemistries performed comparably in quantification of genes and isoforms. The AVITI displayed a marginally lower error rate for long reads, with fewer chemistry-specific errors and a higher mutation detection rate. Conclusion These results point to the potential of the AVITI platform as a competitive candidate in high-throughput short read sequencing analyses when juxtaposed with the Illumina NextSeq 550.
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Zee, Alexander, Dori Zhi Qian Deng, Matthew Adams, et al. "Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2." Genome Research, November 9, 2022, gr.277031.122. http://dx.doi.org/10.1101/gr.277031.122.
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High-throughput short-read sequencing has taken on a central role in research and diagnostics. Hundreds of different assays exist today to take advantage of Illumina short-read sequencers, the predominant short-read sequencing technology available today. Although other short-read sequencing technologies exist, the ubiquity of Illumina sequencers in sequencing core facilities, and the high capital costs of these technologies have limited their adoption. Among a new generation of sequencing technologies, Oxford Nanopore Technologies (ONT) holds a unique position because the ONT MinION, an error-prone long-read sequencer, is associated with little to no capital cost. Here we show that we can make short-read Illumina libraries compatible with the ONT MinION by using the R2C2 method to circularize and amplify the short library molecules. This results in longer DNA molecules containing tandem repeats of the original short library molecules. This longer DNA is ideally suited for the ONT MinION, and after sequencing, the tandem repeats in the resulting raw reads can be converted into high-accuracy consensus reads with similar error rates to that of the Illumina MiSeq. We highlight this capability by producing and benchmarking RNA-seq, ChIP-seq, as well as regular and target-enriched Tn5 libraries. We also explore the use of this approach for rapid evaluation of sequencing library metrics by implementing a real-time analysis workflow.
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Baumann, Alexandra A., Lisanne I. Knol, Marie Arlt, et al. "Long-read genome and RNA sequencing resolve a pathogenic intronic germline LINE-1 insertion in APC." npj Genomic Medicine 10, no. 1 (2025). https://doi.org/10.1038/s41525-025-00485-5.
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Abstract Familial adenomatous polyposis (FAP) is caused by pathogenic germline variants in the tumor suppressor gene APC. Confirmation of diagnosis was not achieved by cancer gene panel and exome sequencing or custom array-CGH in a family with suspected FAP across five generations. Long-read genome sequencing (PacBio), short-read genome sequencing (Illumina), short-read RNA sequencing, and further validations were performed in different tissues of multiple family members. Long-read genome sequencing resolved a 6 kb full-length intronic insertion of a heterozygous LINE-1 element between exons 7 and 8 of APC that could be detected but not fully resolved by short-read genome sequencing. Targeted RNA analysis revealed aberrant splicing resulting in the formation of a pseudo-exon with a premature stop codon. The variant segregated with the phenotype in several family members allowing its evaluation as likely pathogenic. This study supports the utility of long-read DNA sequencing and complementary RNA approaches to tackle unsolved cases of hereditary disease.
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Zhao, Wenxuan, Wei Zeng, Bo Pang, et al. "Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing." Frontiers in Microbiology 14 (May 15, 2023). http://dx.doi.org/10.3389/fmicb.2023.1179966.
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IntroductionGenome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time.MethodsIn this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q &gt; 10) of 98.9%.Results and DiscussionFor a 5-Mb-long bacterial genome, finished genome sequences with accuracy of &gt;99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.
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Fang, Li, Charlly Kao, Michael V. Gonzalez, et al. "LinkedSV for detection of mosaic structural variants from linked-read exome and genome sequencing data." Nature Communications 10, no. 1 (2019). http://dx.doi.org/10.1038/s41467-019-13397-7.
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AbstractLinked-read sequencing provides long-range information on short-read sequencing data by barcoding reads originating from the same DNA molecule, and can improve detection and breakpoint identification for structural variants (SVs). Here we present LinkedSV for SV detection on linked-read sequencing data. LinkedSV considers barcode overlapping and enriched fragment endpoints as signals to detect large SVs, while it leverages read depth, paired-end signals and local assembly to detect small SVs. Benchmarking studies demonstrate that LinkedSV outperforms existing tools, especially on exome data and on somatic SVs with low variant allele frequencies. We demonstrate clinical cases where LinkedSV identifies disease-causal SVs from linked-read exome sequencing data missed by conventional exome sequencing, and show examples where LinkedSV identifies SVs missed by high-coverage long-read sequencing. In summary, LinkedSV can detect SVs missed by conventional short-read and long-read sequencing approaches, and may resolve negative cases from clinical genome/exome sequencing studies.
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Gong, Binsheng, Dan Li, Paweł P. Łabaj, et al. "Targeted DNA-seq and RNA-seq of Reference Samples with Short-read and Long-read Sequencing." Scientific Data 11, no. 1 (2024). http://dx.doi.org/10.1038/s41597-024-03741-y.
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AbstractNext-generation sequencing (NGS) has revolutionized genomic research by enabling high-throughput, cost-effective genome and transcriptome sequencing accelerating personalized medicine for complex diseases, including cancer. Whole genome/transcriptome sequencing (WGS/WTS) provides comprehensive insights, while targeted sequencing is more cost-effective and sensitive. In comparison to short-read sequencing, which still dominates the field due to high speed and cost-effectiveness, long-read sequencing can overcome alignment limitations and better discriminate similar sequences from alternative transcripts or repetitive regions. Hybrid sequencing combines the best strengths of different technologies for a more comprehensive view of genomic/transcriptomic variations. Understanding each technology’s strengths and limitations is critical for translating cutting-edge technologies into clinical applications. In this study, we sequenced DNA and RNA libraries of reference samples using various targeted DNA and RNA panels and the whole transcriptome on both short-read and long-read platforms. This study design enables a comprehensive analysis of sequencing technologies, targeting protocols, and library preparation methods. Our expanded profiling landscape establishes a reference point for assessing current sequencing technologies, facilitating informed decision-making in genomic research and precision medicine.