The Data Preprocessing workflow

Summary

This repository is based off JGI's metatranscriptomic analysis package for transcriptomic reads and uses SAMTOOLS (1.15) in the pipeline readCov_metaTranscriptome_2k20.pl to generate a tab separated read count file from BAM and GFF files.

Running Workflow in Cromwell

Description of the files:

• .wdl file: the WDL file for workflow definition
• .json file: the example input for the workflow
• .conf file: the conf file for running Cromwell.
• .sh file: the shell script for running the example workflow

The Docker image and Dockerfile can be found here

dongyingwu/rnaseqct:1.1

Input files

The inputs for this workflow are as follows:

1. project name
2. BAM File
3. GFF File
4. (optional) Map file
5. (optional) RNA type

{
    "readcount.proj_id":"nmdc:xxxxxxx",
    "readcount.bam": "./test_files/nmdc_xxxxxxx_pairedMapped_sorted.bam",
    "readcount.gff": "./test_files/nmdc_xxxxxxx_functional_annotation.gff",
    "readcount.rna_type": "aRNA",
    "readcount.map": "./test_files/mapfile.map"
}

The map file connects the naming schemes between the GFF and BAM files. If the naming scheme is the same, the map file can either be generated automatically if none is specified, or user can make a tsv with two columns of the names from the GFF file. The RNA type inputs are include nothing, aRNA, or non_stranded_RNA, which are transformed to script inputs (default), -aRNA yes, or -non_stranded yes, respectively. This is the explanation from the script itself:

    -aRNA: yes (means use antisense reads during counting,default no)
    -non_stranded: yes (for cDNA input, default no, override "-aRNA yes" if "-non_stranded yes")

Script

The script is called as such within the WDL.

  readCov_metaTranscriptome_2k20.pl  \
      -b ~{bam} \       # input BAM
      -m ~{map} \       # map file auto generated or user upload
      -g ~{gff} \       # input GFF
      -o ~{out} \       # prefix for output files (project ID)
      ~{rna_type}       # left blank, '-aRNA yes', or '-non_stranded yes'

Output files

The output will have one directory named by prefix project name and a bunch of output files, including statistical numbers, status log, and run information.

The main read count table output is named by prefix.readcount.

|-- nmdc_xxxxxxx.readcount
|-- nmdc_xxxxxxx.readcount.intergenic
|-- nmdc_xxxxxxx.readcount.Stats.log
|-- nmdc_xxxxxxx_readcount.info

Description of IMG metatranscriptome data file.

IMG provides expression values and read counts for gene features predicted on the contigs, be it self-assembly of metatranscriptome or another dataset to which the metatranscriptome reads were mapped. Expression values are computed as mean and median per-base coverage of the sequence within the coordinates of the feature.

Since JGI generally generates stranded libraries, expression values and read counts for two strands are computed and reported separately. These values are NOT expression values and read counts of direct and reverse strand of the contig; instead these are expression values and read counts of the predicted feature (i. e. reads generated for the same strand on which the feature was predicted) and of the opposite strand of the predicted feature. Essentially this "expected" read coverage (in a sense of being generated from the strand that we expect to be expressed) and "unexpected" read coverage (i. e. generated from the strand that we did not expect to be expressed based on structural annotation of the sequence). For obvious reasons, some of the "unexpected" coverage is the result of imperfect structural annotation, which is not uncommon for short contigs in metaT self assembly.

Specific columns in the file:

Column	Description
img_gene_oid	gene_oid of the gene for which expression is counted
img_scaffold_oid	scaffold/contig id on which the gene has been predicted
locus_tag	another gene id of the gene for which expression is counted; this is included because all genomes and some metagenomes and metatranscriptomes used as references have both gene oids and locus tags, while others don't
scaffold_accession	another identifier of scaffold/contig on which the gene has been predicted
strand	strand on which the gene has been predicted
locus_type	type of the gene; for example CDS (protein-coding gene), tRNA, rRNA, tmRNA, etc.
length	length of the gene for which expression is counted
reads_cnt	number of reads mapped on the same strand as predicted gene within the coordinates of the gene
mean	mean expression of the predicted gene, i. e. mean per-base coverage of the strand on which the gene was predicted within the coordinates of the predicted gene
median	median expression of the predicted gene, i. e. median per-base coverage of the strand on which the gene was predicted within the coordinates of the predicted gene
stdev	standard deviation of the expression of the predicted gene
reads_cntA	number of reads mapped to the opposite strand of the predicted gene within the coordinates of the gene
meanA	mean expression of the opposite strand of the predicted gene, i. e. mean per-base coverage of the strand opposite to that on which the gene was predicted within the coordinates of the predicted gene
medianA	median expression of the opposite strand of the predicted gene, i. e. median per-base coverage of the strand opposite to that on which the gene was predicted within the coordinates of the predicted gene
stdevA	standard deviation of the expression of the opposite strand of the predicted gene