Note that the original quality scores are kept in the OQ field of co-cleaned BAM files. This step also increases the accuracy of downstream variant calling algorithms. This step adjusts base quality scores based on detectable and systematic errors.
Base Quality Score RecalibrationĪ base quality score recalibration (BQSR) step is then performed using BaseRecalibrator. Misalignment of indel mutations, which can often be erroneously scored as substitutions, reduces the accuracy of downstream variant calling steps. This step locates regions that contain misalignments across BAM files, which can often be caused by insertion-deletion (indel) mutations with respect to the reference genome. Local realignment of insertions and deletions is performed using IndelRealigner. Both steps of this process are implemented using GATK. the tumor BAM and normal tissue BAM) associated with the same patient. Co-cleaning is performed as a separate pipeline as it uses multiple BAM files (i.e. The alignment quality is further improved by the Co-cleaning workflow. Step 1: Converting BAMs to FASTQs with Biobambam - biobambam2 2.0.54 Note that version numbers may vary in files downloaded from the GDC Portal due to ongoing pipeline development and improvement. Submitted Unaligned Reads or Submitted Aligned ReadsĭNA-Seq Alignment Command Line Parameters Reference sequences used by the GDC can be downloaded here. Ten types of human viral genomes are included: human cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus (HIV), human herpes virus 8 (HHV-8), human T-lymphotropic virus 1 (HTLV-1), Merkel cell polyomavirus (MCV), Simian vacuolating virus 40 (SV40), and human papillomavirus (HPV). Decoy viral sequences are included in the reference genome to prevent reads from aligning erroneously and attract reads from viruses known to be present in human samples. Reference GenomeĪll alignments are performed using the human reference genome GRCh38.d1.vd1. Duplicate reads, which may persist as PCR artifacts, are then flagged to prevent downstream variant call errors. Otherwise BWA-aln is used.Įach read group is aligned to the reference genome separately and all read group alignments that belong to a single aliquot are merged using Picard Tools SortSam and MergeSamFiles. BWA-MEM is used if mean read length is greater than or equal to 70 bp. Read groups are aligned to the reference genome using one of two BWA algorithms. Alignment WorkflowĭNA-Seq analysis begins with the Alignment Workflow. Note that this filtering step is distinct from trimming reads using base quality scores. Reads that failed the Illumina chastity test are removed. Prior to alignment, BAM files that were submitted to the GDC are split by read groups and converted to FASTQ format. An aggregation pipeline incorporates variants from all cases in one project into a MAF file for each pipeline.ĭNA-Seq analysis is implemented across six main procedures: Somatic-caller-identified variants are then annotated. Four different variant calling pipelines are then implemented separately to identify somatic mutations. The first pipeline starts with a reference alignment step followed by co-cleaning to increase the alignment quality. Somatic variants are identified by comparing allele frequencies in normal and tumor sample alignments, annotating each mutation, and aggregating mutations from multiple cases into one project file. The GDC DNA-Seq analysis pipeline identifies somatic variants within whole exome sequencing (WXS) and whole genome sequencing (WGS) data.
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Tumor-Only Variant Call Command-Line Parameters.DNA-Seq Co-Cleaning Command Line Parameters.DNA-Seq Alignment Command Line Parameters.fa-file-text Download PDF /Data/PDF/Data_UG.pdfīioinformatics Pipeline: DNA-Seq Analysis.Bioinformatics Pipeline: Protein Expression.Bioinformatics Pipeline: Methylation Liftover Pipeline.Bioinformatics Pipeline: Copy Number Variation Analysis.Bioinformatics Pipeline: miRNA Analysis.Bioinformatics Pipeline: DNA-Seq Analysis.fa-file-text Download PDF /Data_Transfer_Tool/PDF/Data_Transfer_Tool_UG.pdf.Data Transfer Tool Command Line Documentation.fa-file-text Download PDF /Data_Submission_Portal/PDF/Data_Submission_Portal_UG.pdf.Before Submitting Data to the GDC Portal.fa-file-text Download PDF /Data_Portal/PDF/Data_Portal_UG.pdf.fa-file-text Download PDF /API/PDF/API_UG.pdf.Appendix C: Format of Submission Queries and Responses.
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