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(How to) Create a snippet of reads corresponding to a genomic interval

shleeshlee CambridgeMember, Broadie, Moderator Posts: 595 admin
edited December 2015 in Tutorials

Tools involved

Prerequisites

  • Installed GATK tools
  • Reference genome
  • Coordinate-sorted, aligned and indexed BAM

Download example data

  • Use the advanced tutorial bundle's human_g1k_v37_decoy.fasta as reference
  • tutorial_6517.tar.gz contains four files: 6517_2Mbp_input.bam and .bai covering reads aligning to 10:90,000,000-92,000,000 and 6517_1Mbp_output.bam and .bai covering 10:91,000,000-92,000,000

Related resources


Create a snippet of reads corresponding to a genomic interval using PrintReads

PrintReads merges or subsets sequence data. The tool automatically applies MalformedReadFilter and BadCigarFilter to filter out certain types of reads that cause problems for downstream GATK tools, e.g. reads with mismatching numbers of bases and base qualities or reads with CIGAR strings containing the N operator.

  • To create a test snippet of RNA-Seq data that retains reads with Ns in CIGAR strings, use -U ALLOW_N_CIGAR_READS.

Subsetting reads corresponding to a genomic interval using PrintReads requires reads that are aligned to a reference genome, coordinate-sorted and indexed. Place the .bai index in the same directory as the .bam file.

java -Xmx8G -jar /path/GenomeAnalysisTK.jar \
    -T PrintReads \ 
    -R /path/human_g1k_v37_decoy.fasta \ #reference fasta
    -L 10:91000000-92000000 \ #desired genomic interval chr:start-end
    -I 6517_2Mbp_input.bam \ #input
    -o 6517_1Mbp_output.bam 

This creates a subset of reads from the input file, 6517_2Mbp_input.bam, that align to the interval defined by the -L option, here a 1 Mbp region on chromosome 10. The tool creates two new files, 6517_1Mbp_output.bam and corresponding index 6517_1Mbp_output.bai.

  • For paired reads, the tool does not account for reads whose mate aligns outside of the defined interval. To filter these lost mate reads, use RevertSam's SANITIZE option.

To process large files, also designate a temporary directory.

    TMP_DIR=/path/shlee #sets environmental variable for temporary directory

Post edited by shlee on
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