SNPCallingPipeline

This is a SNP calling tool, which implements read mapping from three different algorithms (BWA, LAST and Novoalign) and evaluates their output. This pipeline currently is only suitable for bacterial genomes.

SNP filtering pipeline by Julio Diaz Caballero @ Guttman lab.

Install

• Download the SNPCallingPipeline
  $ wget https://github.com/DSGlab/SNPCallingPipeline/raw/master/SNPCallingPipeline.jar
• Download the sample configuration file
  $ wget https://github.com/DSGlab/SNPCallingPipeline/raw/master/conf.txt
• Test

$ java -jar SNPCallingPipeline.jar

SNP Calling Pipeline v. 1.12
Questions? [email protected]

Usage:	java -jar SNPCallingPipeline ANALYSIS_NAME [config file]

Setup:		datachecker		Checks naming of input files
Step 0:		scinetjobcreator	Create jobs to be used in scinet's niagara
Step 1:		gethqsnps		Get list of High quuality (confidence) SNPs
Optional step:	getintraclonalsnps	Remove SNPs that are fixed between the isolates and the reference
Step 2:		snpchecker		Gets raw calls at the SNP positions in all the isolates
Step 3:		snpfilter		Filters raw SNP calls
Step 4:		createalignment		Creates alignment based on filtered SNP calls

Setup:

Required Files

• Paired-End sequencing reads in the format: \<id>_1.fq \<id>_2.fq (id is identical as described in the id list).
  
• Reference headers in the format: \<refName>\_\<repliconType>\_\<repliconNum> (e.g. pao1_chromosome_1).
• File including list of strain names (One strain name per line).

Index Reference files

Index BWA

Example: bwa index reference.fa

Index LAST

Example: lastdb reference_LAST reference.fa

Index NOVOALIGN

Example: novoindex reference_NOVOALIGN reference.fa

Strain List File

One strain per line.

strainA
strainB
strainC
strainD

Configuration File:

SETTTINGS FOR scinetojobcreator STEP

• REF_FILE - The reference file in fasta format. Must include complete path to file.
• ID_LIST_FILE - The file including the IDs of all the strains to be taken into acccount (one per line). Must include complete path to file.
• ALIGNER - The name of the aligner to be taken into account for the second step (options: "BWA","LAST","NOVOALIGN").
• INPUT_DIR - The directory which includes the sequencing reads.
• ALIGNMENT_DIR - The directory where the alignment files will be saved.
• JOBS_DIR - The directory where the bash jobs will be saved.
• INCLUDE_QUAKE - TRUE if pipeline is set to include non-quality controlled reads (FALSE otherwise).
• INCLUDE_BWA - TRUE if 'BWA' alignment is required (FALSE not supported at the moment).
• INCLUDE_LAST - TRUE if 'LAST' alignment is required (FALSE not supported at the moment).
• INCLUDE_NOVOALIGN - TRUE if 'NOVOALIGN' alignment is required (FALSE not supported at the moment).
• INCLUDE_BOWTIE2 - TRUE if 'BWA' alignment is required (FALSE is the only option supported at the moment).
• BWA_REF - The
• NOVOALIGN_REF -
• LAST_REF -
• BOWTIE_REF -
• BWA_PATH - Path to the 'BWA' executable.
• LAST_PATH - Path to the 'LAST' executable.
• NOVOALIGN_PATH - Path to the 'NOVOALIGN' executable.
• SAMTOOLS_PATH - Path to the 'SAMTOOLS' executable.
• BCFTOOLS_PATH - Path to the 'BCFTOOLS' executable.

SETTINGS FOR gethqsnps STEP

• HQ_SNP_LIST_OUTPUT - The output file of this step. Must include complete path to file.
• HQ_SNP_WORKING_DIR - The directory where the alignment results are stored.
• HQ_SNP_QUALITY - The minimum quality score required to be considered a HQ SNP.
• HQ_SNP_DEPTH - The minimum depth required to be considered a HQ SNP.
• HQ_SNP_DIST_TO_CONTIG_END - The minimum distance to the end of the replicon required to be considered a HQ SNP.
• HQ_SNP_READ_BALANCE - The minimum number of reads required from both reverse and forward directions to be considered a HQ SNP.
• HQ_SNP_REF_TO_ALT_BALANCE - The minimum reference to alternative call ratio required to be considered a HQ SNP.
• HQ_SNP_CLUSTER_SIZE - The required distance between other SNPs to be considered a HQ SNP.
• HQ_SNP_REQUIRED_NUM - The minimum number of aligners that call the SNP to be considered a HQ SNP (Currently only '3' is supported).
• INCLUDE_PRE_QC - TRUE if pipeline is set to include non-quality controlled reads (FALSE otherwise).

SETTINGS FOR getintraclonalsnps STEP

• INTRA_SNP_INPUT - The output file from the gethqsnps STEP. Must include complete path to file.
• INTRA_SNP_WORKING_DIR - The directory where the alignment results are stored
• INTRA_SNP_OUTPUT - The output file of this step.

SETTINGS FOR snpchecker STEP

• SNP_CHECKER_INPUT - The output file from the gethqsnps (or getintraclonalsnps) STEP. Must include complete path to file.
• SNP_CHECKER_WORKING_DIR - The directory where the alignment results are stored
• SNP_CHECKER_OUTPUT - The output file of this step.

SETTINGS FOR snpfilter STEP

• SNP_FILTER_INPUT - The output file from the snpchecker STEP. Must include complete path to file.
• SNP_FILTER_OUTPUT - The output file of this step.
• SNP_FILTER_MIN_GOOD_BALANCE - The minimum number of reads required from both reverse and forward directions to be considered a SNP.
• SNP_FILTER_MIN_GOOD_CALL_RATIO - The minimum ratio required between a reference and alternative call to be considered a SNP.
• SNP_FILTER_MIN_GOOD_QUALITY - The minimum quality score required to be considered a SNP.

SETTINGS FOR createalignment STEP

• CREATE_ALIGN_INPUT - The output file from the snpfilter STEP. Must include complete path to file.
• CREATE_ALIGN_OUTPUT - The output alignment file in fasta.
• CREATE_ALIGN_LIST_OUTPUT - The output file including the positions where SNPs were detected.

Easy Guide

Setup

1. Create [strain list file](###Strain List File).
2. Have a reference sequences and paired-end sequencing reads in the [required format](###Required Files).
3. [Index](###Index Reference files) the reference.
4. Modify [configuration file](###Configuration File) to match your criteria.
5. Check that setup was completed

$ java -jar SNPCallingPipeline.jar datachecker conf.txt

SNP Calling Pipeline v. 1.12
Questions? [email protected]

Usage:	java -jar SNPCallingPipeline ANALYSIS_NAME [config file]

Checking input files.

0 error(s) found in the reference or seq read files
0 error(s) found in the path to the software

6. Create jobs

$ java -jar SNPCallingPipeline.jar scinetjobcreator conf.txt

SNP Calling Pipeline v. 1.12
Questions? [email protected]

Usage:	java -jar SNPCallingPipeline ANALYSIS_NAME [config file]

Starting Create_Alignment analysis.
	Printing task for isolate:	strainA
	Printing task for isolate:	strainB
	Printing task for isolate:	strainC
	Printing task for isolate:	strainD

This created a number of bash scripts in the jobs directory

$ ls jobsDirectory

aligner0.sh     bwa-strainA.sh       bwa-strainB.sh       bwa-strainC.sh       bwa-strainD.sh
last-strainA.sh      last-strainB.sh      last-strainC.sh       last-strainD.sh
novoalign-strainA.sh novoalign-strainB.sh novoalign-strainC.sh novoalign-strainD.sh

The bwa*.sh, novoalign*.sh, and last*.sh files can be executed in the terminal as usual

$ bash bwa-strainA.sh
$ bash bwa-strainB.sh
$ bash bwa-strainC.sh
$ bash bwa-strainD.sh
$ bash last-strainA.sh
$ bash last-strainB.sh
$ bash last-strainC.sh
$ bash last-strainD.sh
$ bash novoalign-strainA.sh
$ bash novoalign-strainB.sh
$ bash novoalign-strainC.sh
$ bash novoalign-strainD.sh

OR the aligner?.sh bash scripts can be submitted to niagara in scinet.

$ sbatch aligner0.sh

Wait for the scripts to finsh.
6. Run gethqns step

$ java -jar SNPCallingPipeline.jar gethqsnps conf.txt

7. Run getintraclonalsnps step

$ java -jar SNPCallingPipeline.jar getintraclonalsnps conf.txt

8. Run snpchecker step

$ java -jar SNPCallingPipeline.jar snpchecker conf.txt

9. Run snpfilter step

$ java -jar SNPCallingPipeline.jar snpfilter conf.txt

10. Run createalignment step

$ java -jar SNPCallingPipeline.jar createalignment conf.txt

11. Results

$ ls outputDirectory

snp_alignment.fa
snp_list.fa

• snp_alignment.fa - all positions with HQ SNPs with the proper call
• snp_list.fa - info about every polymorphic position