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megSAP - DNA long-read analysis (single sample)

Basics

Single sample long-read DNA analysis is performed using the analyze_longread.php script.
Please have a look at the help using:

> php megSAP/src/Pipelines/analyze_longread.php --help

The main parameters that you have to provide are:

  • folder - The sample folder, which contains the the FASTQ files as produced by bcl2fastq2.
  • name - The sample name, which must be a prefix of the FASTQ files.
  • steps - Analysis steps to perform. Please use ma,vc,sv,re,an,db to perform mapping, variant calling (SNV/InDels, CNVs, SVs and REs), annotation and database import.
  • system - The processing system INI file.

Running an analysis

The analysis pipeline assumes that that all data to analyze (FastQ files) resides in a sample folder. If that is the case, the whole analysis is performed with one command, for example like this:

php megSAP/src/Pipelines/analyze_longread.php -folder Sample_NA12878_01 -name NA12878_01 -system SQK-114.ini -steps ma,vc,cn,sv,re,an

In the example above, the configuration of the pipeline is done using the SQK-114.ini file, which contains all necessary information (see processing system INI file).

Tools used in this analysis pipeline

The following tools are used for mapping and calling of small variants and annotation of small variants:

step tool comments
mapping minimap2
mapping - extract methylation info modkit
variant calling - calling of SNVs and InDels clair3
variant calling - decompose complex variants vcfallelicprimitives
variant calling - break multi-allelic variants vcfbreakmulti
variant calling - left-normalization of InDels VcfLeftNormalize
annotation VEP

CNV calling and annotation is performed using these tools:

step tool comments
CNV calling ClinCNV
annotation - general BedAnnotateFromBed Several data sources are annotated using this tool.
annotation - gene information CnvGeneAnnotation
annotation - overlapping pathogenic CNVs from NGSD NGSDAnnotateCNV

SV calling and annotation is performed using these tools:

step tool comments
SV calling Sniffles
annotation - gene information BedpeGeneAnnotation
annotation - matching SVs from NGSD BedpeAnnotateCounts
annotation - breakpoint density from NGSD BedpeAnnotateBreakpointDensity

RE calling using these tools:

step tool comments
RE calling Straglr

Phasing is performed using these tools:

step tool comments
phasing longphase

A complete list of all tools and databases used in megSAP including version and when they were last updated can be found here.

Performance

Performance benchmarks of the the megSAP pipeline can be found here.

Output

After the analysis, these files are created in the output folder:

  1. mapped reads in BAM format
  2. a variant list in VCF format
  3. a variant list in GSvar format
  4. QC data in qcML format, which can be opened with a web browser