Best practices for variant calling in clinical sequencing

Table 2 Key components of NGS analysis and a list of exemplar tools. Most clinical sequencing pipelines will employ a single read aligner (e.g., BWA-MEM) and mark duplicates with one algorithm (e.g., Picard). However, multiple tools for collecting sequencing metrics and performing sample QC may be employed to meet the needs of the laboratory. For variant calling, it is recommended that pipelines incorporate 2–3 tools for each class of variant to maximize detection sensitivity. See the relevant section of this review for recommendations specific to each variant class

Strategy	Variant callers
Alignment and pre-processing
Read alignment	BWA-MEM [25], Bowtie 2 [26], minimap2 [27], Novoalign
Marking duplicates	Picard tools [28], Sambamba [29], SAMBLASTER [30]
BAM file creation	Samtools [31], GATK [19]
Sequencing metrics	BEDTools [32], Picard tools [28], QualiMap 2 [33]
Sample quality control	KING [34], VerifyBamID [35]
Variant calling
Inherited SNVs/indels	FreeBayes [36], GATK HaplotypeCaller [19], Platypus [20], Samtools/BCFtools [37]
Somatic mutations	deepSNV [38], MuSE [39], MuTect2 [40], SomaticSniper [41], Strelka2 [42], VarDict [43], VarScan2 [44]
Copy number variants	cn.MOPS [45], CONTRA [46], CoNVEX [47], ExomeCNV [48], ExomeDepth [49], XHMM [50]
Structural variants	DELLY [51], Lumpy [52], Manta [53], Pindel [54], SVMerge [55]
Gene fusions (RNA-seq)	fusionCatcher [56], fusionMap [57], mapSplice [58], SOAPfuse [59], STAR-Fusion [60], TopHat-Fusion [61]
Variant review/storage
Visualization and review	Artemis [62], Integrative Genomics Viewer [63]
VCF/BCF file manipulation	BCFtools [37]

BAM binary alignment/map, SNV single nucleotide variant, VCF variant call format, BCF binary variant call format

ISSN: 1756-994X