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 usema,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.
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).
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 benchmarks of the the megSAP pipeline can be found here.
After the analysis, these files are created in the output folder:
- mapped reads in BAM format
- a variant list in VCF format
- a variant list in GSvar format
- QC data in qcML format, which can be opened with a web browser