MEND QC

Calculates the number of Mapped Exonic Non-Duplicate (MEND) reads in a bam file containing RNA-Seq data.

Overview

samblaster is used to mark duplicates and sambamba is used to sort. Then RSeqQC calculates the reads distribution over exons skipping reads marked qc_failed, PCR duplicate, Unmapped, Non-primary (or secondary). The MEND qc script parseReadDist.R estimates the number of MEND reads by counting tags in CDS exons, 5' UTR exons and 3' UTR exons and multiplying by reads per tag.

Output

• readDist.txt: The output of RSeqQC read_distribution.py (~1kb)
• bam_mend_qc.tsv: MND, MEND and treehouse_compendium_qc (PASS/FAIL)
• bam_mend_qc.json: Same as bam_mend_qc.tsv but in json format
• sortedByCoord.md.bam: BAM with duplicates marked sorted by coordinate
• sortedByCoord.md.bam.bai: Index for sortedByCoord.md.bam

Running

via Docker:

docker run --rm \
  -v <path to bam file>:/inputs/sample.bam \
  -v <path to output>:/outputs \
  -v <path to tmp space>:/tmp \
  ucsctreehouse/bam-mend-qc \
    /inputs/sample.bam \
    /outputs

Optionally, specify a bed file as the third argument (after "/outputs"). The bed file needs to be formatted as specified by RSeQC. If not specified, is uses a bed file containing exon definitions specified by GENCODE_v23_basic in hg38 coordinates.

Note: Intermediate bam files are created under /tmp within the docker container.

Directly:

run.sh <path to bam> <path to output folder> <optionally: path to bed file>

NOTE: See Dockerfile for installation of required libraries

Example expected stdout

Sorting by name...
Marking duplicates...
samblaster: Version 0.1.24
samblaster: Inputting from stdin
samblaster: Outputting to stdout
samblaster: Loaded 195 header sequence entries.
samblaster: Marked 1 of 999 (0.10%) read ids as duplicates using 1340k memory in 0.005S CPU seconds and 0S wall tim
e.
Sorting by coordinate...
Writing sorted chunks to temporary directory...
[==============================================================================]
Counting reads...
processing /ref/hg38_GENCODE_v23_basic.bed ... Done
processing /tmp/TEST.bam.sortedByCoord.md.bam ... Finished

[1] "analyzing /data/readDist.txt"
Read 3 items