Academic literature on the topic 'Reference protein-coding alignments'

EzEditor is a Java-based molecular sequence editor allowing manipulation of both DNA and protein sequence alignments for phylogenetic analysis. It has multiple features optimized to connect initial computer-generated multiple alignment and subsequent phylogenetic analysis by providing manual editing with reference to biological information specific to the genes under consideration. It provides various functionalities for editing rRNA alignments using secondary structure information. In addition, it supports simultaneous editing of both DNA sequences and their translated protein sequences for protein-coding genes. EzEditor is, to our knowledge, the first sequence editing software designed for both rRNA- and protein-coding genes with the visualization of biologically relevant information and should be useful in molecular phylogenetic studies. EzEditor is based on Java, can be run on all major computer operating systems and is freely available from http://sw.ezbiocloud.net/ezeditor/.

ABSTRACT We report here an update of the Botrytis cinerea strains B05.10 and T4 genomes, as well as an automated preliminary gene structure annotation. High-coverage de novo assemblies and reference-based alignments led to a correction of wrong base calls, elimination of sequence gaps, and improved joining of contigs. The new assemblies have substantially lower numbers of scaffolds and a concomitant increase in the N 50 .The list of protein-coding genes was generated using the evidence-driven gene predictor Augustus, with expressed sequence tag evidence and RNA-Seq data as input.

Abstract Summary Diverse traits have evolved through cis-regulatory changes in genome sequence that influence the magnitude, timing and cell type-specificity of gene expression. Advances in high-throughput sequencing and regulatory genomics have led to the identification of regulatory elements in individual species, but these genomic regions remain difficult to align across taxonomic orders due to their lack of sequence conservation relative to protein coding genes. The groundwork for tracing the evolution of regulatory elements is provided by the recent assembly of hundreds of genomes, the generation of reference-free Cactus multiple sequence alignments of these genomes, and the development of the halLiftover tool for mapping regions across these alignments. We present halLiftover Post-processing for the Evolution of Regulatory Elements (HALPER), a tool for constructing contiguous regulatory element orthologs from the outputs of halLiftover. We anticipate that this tool will enable users to efficiently identify orthologs of regulatory elements across hundreds of species, providing novel insights into the evolution of traits that have evolved through gene expression. Availability and implementation HALPER is implemented in python and available on github: https://github.com/pfenninglab/halLiftover-postprocessing. Supplementary information Supplementary data are available at Bioinformatics online.

Abstract The recent extensive application of next-generation sequencing has led to the rapid accumulation of multiple types of data for functional DNA elements. With the advent of precision medicine, the fine-mapping of risk loci based on these elements has become of paramount importance. In this study, we obtained the human reference genome (GRCh38) and the main DNA sequence elements, including protein-coding genes, miRNAs, lncRNAs and single nucleotide polymorphism flanking sequences, from different repositories. We then realigned these elements to identify their exact locations on the genome. Overall, 5%–20% of all sequence element locations deviated among databases, on the scale of kilobase-pair to megabase-pair. These deviations even affected the selection of genome-wide association study risk-associated genes. Our results implied that the location information for functional DNA elements may deviate among public databases. Researchers should take care when using cross-database sources and should perform pilot sequence alignments before element location-based studies.

Abstract Abstract 300 Background: Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of diseases characterized by clonal expansion of malignant T-cells in the skin. The two predominant clinical forms of CTCL are mycosis fungoides (MF) and Sezary syndrome (SS). Tumor-stage MF has an unfavorable prognosis with a 10-year survival of approximately 40%. The molecular pathogenesis of CTCL is still basically unknown, although some data suggest that signalling from T-cell receptor (TCR) is a driving force. However, the molecular mechanisms responsible for this activation have not been fully clarified. Methods: Based on the hypothesis that TCR activation may depend, at least in part, on somatic mutations, we have investigated this in a selection of genes belonging to TCR, or related pathways, such as NFkB, JAK/STAT, by means of deep sequencing. A Target Enrichment method using SureSelect system (Agilent) has been used to enrich in exons and regulatory regions of 524 genes belonging to these pathways. DNA from 2 tumoral-MF, 5 erythrodermic-MF and 4 SS patients, both normal and tumoral, were processed and sequenced with Genome Analyzer GA2 (Illumina) (PE-42bp). Sequencing data were first checked by FastQC and aligned to the human reference genome (GRCh37) using BWA and BFAST alignments. Somatic variants were identified using GATK. Thus, SNPs available at dbSNP 135 (hg19) and 1000 Genomes Project were filtered out from VCF output files. The GATK-QUAL field was employed for ranking selected somatic variants. Biological impact predictions for detected variants were obtained from Ensembl Variant Effect Predictor. Putative variants were manually reviewed and validated by capillary sequencing. Immunohistochemical analysis for NFAT, p50, p52 and STAT·p was also performed. qPCR-genotyping for specific variants was performed in a new cohort of 60 CTCL patients including SS and tumoral MFs. Results: Several mutations were found in essential genes belonging to pathways implicated in the Treg and Th17 regulatory pathways, NFkB and JAK/STAT, among others. PLCG1 was found mutated in three samples, two of them sharing the same mutation affecting one of the PLCG1 protein catalytic domains. This mutation was further analyzed by qPCR-genotyping in the new series of patients, being detected in 20% of samples. PLCG mutated cases showed a strong paraffin immunostaining for nuclear NFAT, p50 and p52. Additionally, immunological studies performed by flow cytometry in CTCL cell lines show aberrant coexpression of TH17 and Treg phenotypes. Conclusions: Activation of the TCR in CTCL might be partially dependent on the acquisition of somatic mutations in the coding region of genes known to play an essential role in T-cell differentiation processes and acquisition of TH17 and Treg phenotypes. Especially relevant is the finding that the catalytic domain of PLCG1 is frequently mutated in tumoral MF samples. Disclosures: No relevant conflicts of interest to declare.

Technological advances have allowed improvements in genome reference sequence assemblies. Here, we combined long- and short-read sequence resources to assemble the genome of a female Great Dane dog. This assembly has improved continuity compared to the existing Boxer-derived (CanFam3.1) reference genome. Annotation of the Great Dane assembly identified 22,182 protein-coding gene models and 7,049 long noncoding RNAs, including 49 protein-coding genes not present in the CanFam3.1 reference. The Great Dane assembly spans the majority of sequence gaps in the CanFam3.1 reference and illustrates that 2,151 gaps overlap the transcription start site of a predicted protein-coding gene. Moreover, a subset of the resolved gaps, which have an 80.95% median GC content, localize to transcription start sites and recombination hotspots more often than expected by chance, suggesting the stable canine recombinational landscape has shaped genome architecture. Alignment of the Great Dane and CanFam3.1 assemblies identified 16,834 deletions and 15,621 insertions, as well as 2,665 deletions and 3,493 insertions located on secondary contigs. These structural variants are dominated by retrotransposon insertion/deletion polymorphisms and include 16,221 dimorphic canine short interspersed elements (SINECs) and 1,121 dimorphic long interspersed element-1 sequences (LINE-1_Cfs). Analysis of sequences flanking the 3′ end of LINE-1_Cfs (i.e., LINE-1_Cf 3′-transductions) suggests multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations. Consistent with this conclusion, we demonstrate that a canine LINE-1_Cf element with intact open reading frames can retrotranspose its own RNA and that of a SINEC_Cf consensus sequence in cultured human cells, implicating ongoing retrotransposon activity as a driver of canine genetic variation.

Abstract Hares (genus Lepus) provide clear examples of repeated and often massive introgressive hybridization and striking local adaptations. Genomic studies on this group have so far relied on comparisons to the European rabbit (Oryctolagus cuniculus) reference genome. Here, we report the first de novo draft reference genome for a hare species, the mountain hare (Lepus timidus), and evaluate the efficacy of whole-genome re-sequencing analyses using the new reference versus using the rabbit reference genome. The genome was assembled using the ALLPATHS-LG protocol with a combination of overlapping pair and mate-pair Illumina sequencing (77x coverage). The assembly contained 32,294 scaffolds with a total length of 2.7 Gb and a scaffold N50 of 3.4 Mb. Re-scaffolding based on the rabbit reference reduced the total number of scaffolds to 4,205 with a scaffold N50 of 194 Mb. A correspondence was found between 22 of these hare scaffolds and the rabbit chromosomes, based on gene content and direct alignment. We annotated 24,578 protein coding genes by combining ab-initio predictions, homology search, and transcriptome data, of which 683 were solely derived from hare-specific transcriptome data. The hare reference genome is therefore a new resource to discover and investigate hare-specific variation. Similar estimates of heterozygosity and inferred demographic history profiles were obtained when mapping hare whole-genome re-sequencing data to the new hare draft genome or to alternative references based on the rabbit genome. Our results validate previous reference-based strategies and suggest that the chromosome-scale hare draft genome should enable chromosome-wide analyses and genome scans on hares.

Abstract INTRODUCTION: The cellular iron exporter ferroportin, encoded by the SLC40A1 gene, plays a key role in systemic iron regulation by mediating the absorption of dietary iron from duodenal enterocytes and the release of macrophage iron stores into the plasma. SLC40A1 mutations result in a clinically heterogeneous iron overload disorder exhibiting autosomal dominant transmission. Mutations that impair iron export function result in a classical ferroportin disease phenotype characterized by hyperferritinemia, normal transferrin saturation, and macrophage iron loading, while mutations that impair the regulation of ferroportin by hepcidin result in a non-classical form of disease exhibiting high transferrin saturation and hepatocellular iron loading. Here we report the clinical phenotype of a male patient with a personal and family history of iron overload who was found to harbor a novel SLC40A1 mutation. CLINICAL HISTORY: A 39-year-old male of Italian descent came to clinical attention after laboratory evidence of iron overload was detected at the time of a routine physical exam. Serum ferritin was markedly elevated at 5018 ng/mL, while transferrin iron saturation was within the normal range at 42%. A complete blood count revealed hemoglobin 16.1 g/dL, hematocrit 48.7%, and MCV 96.7 fL. Liver function tests revealed mild elevation of transaminases (AST 50 U/L, ALT 114 U/L). HBV and HCV serologies, as well as an anti-nuclear antibody screen, were negative. Rheumatoid factor, ceruloplasmin, and alpha-fetoprotein were within the normal range. Genetic testing for the HFE C282Y, H63D, and S65C variants was negative. Abdominal ultrasound revealed a somewhat course and echogenic liver. The patient's past medical and surgical histories were non-contributory. Family history was notable for a 63-year-old father with non-HFE hemochromatosis treated by phlebotomy, as well as a possible history of iron overload in the paternal grandmother. There was no known family history of hepatocellular carcinoma. The patient reported consuming ≤ 5 alcoholic beverages per week. Review of systems was negative, and no organomegaly was detected on physical exam. The patient began undergoing phlebotomy approximately every 2 weeks, which now has been well tolerated for almost two years. His most recent ferritin level was within the normal range (328 ng/mL). METHODS: Using genomic DNA extracted from peripheral blood as template, all coding regions and intron-exon boundaries of SLC40A1 were amplified by polymerase chain reaction and analyzed by bidirectional Sanger sequencing. Sequence chromatograms were analyzed using Sequencher software. This study was approved by the Yale University Human Investigation Committee (protocol #010412377). RESULTS: A heterozygous single nucleotide substitution (c.263G>T, encoding p.Arg88Ile) was identified in exon 3 of the SLC40A1 gene (nomenclature per Ensembl reference transcript ENST00000261024). Ferroportin is a predicted multipass transmembrane protein, and Arg88 resides between two predicted transmembrane domains. Protein sequence alignments reveal that amino acid 88 is conserved as an arginine in ferroportin homologs in species as evolutionarily distant as Xenopus laevis and Danio rerio, suggesting that this residue is required for normal ferroportin function. The p.Arg88Ile variant has not been reported in the 1000 Genomes Project or the Exome Aggregation Consortium, demonstrating that it is not a polymorphism in the general population. The mutation predictor algorithms PolyPhen2, SIFT, and MutationTaster all strongly predicted this mutation to be damaging. DISCUSSION: The ferroportin variant detected in this case (p.Arg88Ile) represents the third non-synonymous substitution at ferroportin residue 88 detected in patients with iron overload phenotypes. p.Arg88Gly was identified in a 38-year-old male who became anemic under a phlebotomy program (Cunat S., et al., Clin Chem 2007), while p.Arg88Thr was detected in multiple affected members of a single kindred in whom serial phlebotomy was well tolerated (Bach V, et al. Blood Cells Mol Dis 2006). Collectively, these findings suggest that the particular amino acid substitution at residue 88 may influence the degree of cellular iron sequestration. Future work will assess the effect of the Arg88Ile substitution on ferroportin function. Disclosures No relevant conflicts of interest to declare.

The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.

Rangifer tarandus, known as caribou or reindeer, is a widespread circumpolar species which presents significant variability in their morphology, ecology, and genetics. A genome was sequenced from a male boreal caribou (R. t. caribou) from Manitoba, Canada. Both paired end and Chicago libraries were constructed and sequenced on Illumina platforms. The final assembly consists of approximately 2.205 Gb, and has a scaffold N50 of 11.765 Mb. BUSCO (Benchmarking Universal Single-Copy Orthologs) reconstructed 3820 (93.1%) complete mammalian genes, and genome annotation identified the locations of 33,177 protein-coding genes. An alignment to the bovine genome was carried out, indicating sequence coverage on all bovine chromosomes. A high-quality reference genome will be invaluable for evolutionary research and for conservation efforts for the species. Further information about the genome, including a FASTA file of the assembly and the annotation files, is available on our caribou genome website. Raw sequence data is available at the National Centre for Biotechnology Information (NCBI), under the BioProject accession number PRJNA549927.