SeqQEst

Quality control for next-generation sequencing data

Author: Hua Sun
Version: v1.02

• Updated (2021-03-20)

• Updated (2021-03-01)

  • v1.02 -- Add hlaQC.fq.call_hla.sh

• Previous version

  • Version v1.0: https://github.com/ding-lab/SeqQEst/tree/v1.0
  • Beta version: https://github.com/ding-lab/SeqQEst/tree/beta-version

Introduction

SeqQEst is a tool to estimate single- and multiple-sample sequence quality, to detect swapped, mislabeled, or contaminated samples, and to confirm genetic lineages through consideration of bulk DNA- and RNA-seq data.

Requirements

Install (conda)

Install below tools by "sh setup/setup_tools.sh"

The "setup_tools.sh" will install tools and output config_seqQEst.ini, which will list all of tool locations.

    	JRE v1.8
    	Picard >=v2.17.11
    	Samtools >=v1.5
    	FastQC >=v0.11.9
    	7z >=v15.09
    	Bam-readcount v0.7.4
    	BWA v0.7.17
    	OptiType v1.3.2
    
    	Python 3.x (>=3.7)
	    	Pandas v1.2.1
	    	Matplotlib v3.3.4
	    	Seabornd v0.11.1
		Perl >=v5.18.2

Set database

• Genome *.fa

  • GENCODE GRCh38

  [Note] The pipeline was tested using GENCODE_GRCh38_v29 reference

• Target interval_list form (qc1 - WES coverage)

  • https://gatk.broadinstitute.org/hc/en-us/articles/360035531852-Intervals-and-interval-lists
  • Refer to interval_list/proteinCoding.cds.merged.gencode_hg38_v29.interval_list

• SNP loci (qc2)

  • See folder loci/
  • Note: To obtain the loci information, please contact with author.

• HLA database (qc3)

  • See folder data/
  • Source: https://github.com/FRED-2/OptiType/tree/master/data

Set config

• Set "set_optiType.ini" (set razers3 location)
• Set "config.ini", please refer to config/config.demo.ini
• Please refer to 'setup/setup.sh' output config_seqQEst.ini to set

Input

• BAM

  • WGS
  • WES
  • RNA-Seq

• FASTQ

  • HLA-QC is able to use input as BAM or fastq.gz

• Table of bam/sample info

  • bam.catalog (Form)

    The table uses for running cohort

     ID   DataType   Bam
     TWDE-M1511917 WES /path/PDX/TWDE-M1511917.bam
    

     [Note]
     DataType: WGS/WES/RNA-Seq
    

  • sample.info (Form)

    The table uses for summary of qc2 & qc3

     CaseID	ID	NewID	DataType	Group
    WU12	TWDE242	WU12.WES.N	WES	Human_Normal
    WU59	TWDE4639	WU59.WES.P2	WES	PDX
    

    [Note]
    CaseID: Case ID
    ID: Data ID
    DataType: WGS/WES/RNA-Seq
    Group: Human_Normal/Human_Tumor/PDX
    NewID: If there is no short name then set as '.'
     	
    

Output

• SeqQC

   qc.SeqQC/
   	qc1.seq.summary.merged.out        # summary report
  

• GermlineQC

   qc.germlineQC/
   summary_germlineQC/
   	report.summary_germlineQC.out     # summary report
   	all_of_corAllSamplesPair.tsv
   	export_corr_matrix.tsv
   	report.germlineQC.potential.swap_data.out
   	...
  

• HLA-QC

   qc.hlaQC/
   summary_hlaQC/
   	report.summary_hlaQC.out          # summary report
   	report.hla_with_info.out
  

Usage

sh SeqQEst.sh -c <config.ini> -p <pipelineName> -n <name> -b <bam> -o <outdir>


Tip: If you don’t want to set “contig.ini” repeatedly, then you can set it inside the "SeqQEst.sh".

e.g.
open "SeqQEst.sh" and set 
config="/fullpath/config.ini"

sh SeqQEst.sh -p <pipelineName> -n <name> -b <bam> -o <outdir>

SeqQC (qc1)

# fastqc
sh SeqQEst.sh -p fastqc -n sampleName -b sample.bam -o qc.seqQC

# cov for WES
sh SeqQEst.sh -p tarcov -n sampleName -b sample.bam -o qc.seqQC

# depth (use for WGS)
sh SeqQEst.sh -p meanDepth -n sampleName -b sample.bam -o qc.seqQC

# flagstat & stat
sh SeqQEst.sh -p stat -n sampleName -b sample.bam -o qc.seqQC

# merge seqQC results
sh SeqQEst.sh -p qc1-merge -d qc.seqQC

# qc1 summary and plot
sh SeqQEst.sh -p qc1-summary -m matrix.tsv -f sample.info -o summary_seqQC

GermlineQC (qc2)

# call bamreadcount
sh SeqQEst.sh -p qc2-brc -n sample -b sample.bam -o qc.germlineQC

# qc2 merge
sh SeqQEst.sh -p qc2-merge -d qc.germlineQC/brc -o qc.germlineQC

# qc2 summary
sh SeqQEst.sh -p qc2-summary -f sample.info -m qc2.snp.merged_vaf.table -o summary_germlineQC

# plot for qc2
sh SeqQEst.sh -p qc2-plot -m matrix.tsv -o summary_germlineQC

HLA-QC (qc3)

# qc3 call hla (-t dna/rna) - bam
sh SeqQEst.sh -p qc3-hla -n samplename -t dna -b sample.bam -o qc.hlaQC

# qc3 call hla (-t dna/rna) - fastq
sh SeqQEst.sh -p qc3-hla -n samplename -t dna -1 r1.fastq.gz -2 r2.fastq.gz -o qc.hlaQC


# qc3 merge
sh SeqQEst.sh -p qc3-merge -d qc.hlaQC -o qc.hlaQC

# qc3 summary
sh SeqQEst.sh -p qc3-summary -f sample.info -m qc3.hla.merged.out -o summary_hlaQC

Tutorial: Run multiple samples in cluster

MGI-Server (LSF)
[Note] Three of QCs are independent. Hence you can run all programs at the same time.
	
1. Data Processing
	
	* SeqQC
	> Input  : bam.catalog.table
	> Output : qc.seqQC
	sh worklogs/run.seqQC.fromTable.sh -T ./demo.bam.catalog.table
	
	* GermlineQC
	> Input  : bam.catalog.table
	> Output : qc.germlineQC/brc
	sh worklogs/run.germlineQC.callSNPs.fromTable.sh -T ./demo.bam.catalog.table
	
	* HLA-QC
	> Input  : bam.catalog.table
	> Output : qc.hlaQC
	sh worklogs/run.hlaQC.callHLA.fromTable.sh -T ./demo.bam.catalog.table




2. Summary Report (Table)
	
	* SeqQC
	> Input  : qc.seqQC
	> Output : qc.seqQC/qc1.seq.summary.merged.out
	sh SeqQEst.sh -p qc1-merge -d ./qc.seqQC
	
	> Input  : 1. qc.seqQC/qc1.seq.summary.merged.out
	           2. sample.info 
	> Output : summary_seqQC/report.summary_seqQC.out
	sh SeqQEst.sh -m qc.seqQC/qc1.seq.summary.merged.out -f sample.info -o summary_seqQC
	
	
	* GermlineQC
	> Input  : qc.germlineQC/brc
	> Output : qc.germlineQC/qc2.snp.merged_vaf.table
	sh SeqQEst.sh -p qc2-merge -d qc.germlineQC/brc -o qc.germlineQC
	
	> Input  : 1. qc.germlineQC/qc2.snp.merged_vaf.table
	           2. sample.info 
	> Output : summary_germlineQC/report.summary_germlineQC.out
	sh SeqQEst.sh -p qc2-summary -m qc.germlineQC/qc2.snp.merged_vaf.table -f sample.info -o summary_germlineQC
	
	# plot
	sh SeqQEst.sh -p qc2-plot -m summary_germlineQC/export_corr_matrix.tsv -o summary_germlineQC
	
	
	* HLA-QC
	> Input  : qc.hlaQC
	> Output : qc.hlaQC/qc3.hla.merged.out
	sh SeqQEst.sh -p qc3-merge -d qc.hlaQC -o qc.hlaQC
	
	> Input  : 1. qc.hlaQC/qc3.hla.merged.out
	           2. sample.info
	> Output : summary_hlaQC/report.summary_hlaQC.out
	sh SeqQEst.sh -p qc3-summary -f sample.info -m qc.hlaQC/qc3.hla.merged.out -o summary_hlaQC

Contact

Hua Sun, hua.sun@wustl.edu