pickaxe

Virus discovery pipeline

Overview

Pickaxe identifies potential infectious agents in NGS sequencing reads. It reports on the number of alignments to each virus and metrics such as sequence coverage and average depth. Reads are assembled into contigs for novel virus discovery using search algorithms such as BLAST.

Documentation

This README.md and for more details run pickaxe.pl -h

Installation

• Install annotater and test that it is working.
• Add the pickaxe folder to PATH and pickaxe/lib to PERL5LIB.
• Install the following software. The listed version may not be absolutely required. Other versions were not tested
  • samtools 1.14
  • bowtie2 2.3.3
  • cutadapt 1.18
  • PRINSEQ-lite 0.20.4
  • RepeatMasker open-4.0.7
  • clc_assembler 5.1.1.184548-201811011136 (or megahit 1.2.9)
  • blast+ 2.13.0
  • diamond 2.0.15
  • rapsearch v2.24 (optional)
  • sra-toolkit 2.9.2 (optional)

Test Dataset

This example uses 5000 reads from SRR11074364 which is RNA-Seq of Human RPTE cells infected by the virus BKV. Switch to the t directory and run the following. Depending on your computer, this will take about 1 hour. The test commands run pickaxe with many default parameters and requires the following 1) ref_viruses_rep_genomes bowtie2 index and the ref_viruses_rep_genomes.fa file used to create the index, 2) ref_viruses_rep_genomes blast database, and 3) the NCBI taxonomy database. Note: add --shell parameter to run without submitting a SLURM sbatch job. If you do not have these items installed, see the Docker section below.

pickaxe.pl --rm_default_index SRR11074364_
pickaxe.pl --collate both SRR11074364_
pickaxe.pl --extendcontigs SRR11074364_
pickaxe.pl --stats SRR11074364_

This will generate the following output files:

SRR11074364_.pickaxe/
├── annotator/
├── assembly.ra.tsv
├── assembly.RM.fa
├── assembly.RM.fa.fai
├── extendedcontigs/
├── kv.vrs.hg19.bam
├── okfiles/
├── pickaxe.job
├── pickaxe.job.SRR11074364_.out
├── pickaxe.OK
├── report_BLAST.tsv
├── report_ViralRefSeq.tsv
├── software_versions.tsv
├── SRR11074364_1_5k.fastq -> /path/to/t/SRR11074364_1_5k.fastq
├── SRR11074364_2_5k.fastq -> /path/to/t/SRR11074364_2_5k.fastq
└── unmapped.fq.gz

The description of the files:

• unmapped.fq.gz - 'non-host' reads that survived QC filtering and subtraction against host genomes.
• assembly.RM.fa - raw contigs
• kv.vrs.hg19.bam - bowtie2 alignments to the Viral RefSeq database
• annotator/ - folder containing raw results of annotater
• report_ViralRefSeq.tsv - summarization of the alignments to each virus reference sequence
• report_BLAST.tsv - contig annotation results of the BLAST pipeline found in the BAM file
• extendedcontigs - folder containing extended contigs

Expected results

The report_ViralRefSeq.tsv file should show the following: number of alignments to BKV ~1500 (column 'aligns'), sequence coverage ~87% (column 'seqcov') and average depth ~22 (column 'avgdepth').

The longest contig annotated by the BLAST pipeline as BKV should be ~1200bp (see report_BLAST.tsv).

Docker

You can test pickaxe using the Docker image if you do not have any of the required databases or dependencies installed. First install Docker on your system and build the pickaxe image:

docker build -t pickaxe .

Then enter the t directory and following the instructions in the README.txt