Table 4 ∣.
Substeps in data analysis pipeline
| Step | Software (main option) | Note |
|---|---|---|
| 82.1 | FastQC | QC of raw sequencing read |
| 82.2 | GATK FastqToSam | Convert fastq files to unmapped bam file |
| 82.3 | GATK MarkIlluminaAdapters | Mark sequencing adapters in unmapped bam file |
| 82.4 | GATK SamToFastq; bwa; samtools | Sequence alignment |
| 82.5 | GATK MergeBamAlignment | Merge mapped bam file with unmapped bam file |
| 82.6 | GATK SortSam; GATK SetNmMdAndUqTags | Sort BAM file by coordinate order and fix tag values for NM and UQ |
| 82.7 | GATK MarkDuplicates | Mark duplicate reads to avoid counting non-independent observations |
| 82.8 | GATK BaseRecalibrator | Generate base quality score recalibration model |
| 82.9 | GATK ApplyBQSR | Apply base quality score recalibration model |
| 82.10 | GATK SortSam; GATK BuildBamIndex; md5sum | Sort by coordinate, index and calculate md5 |
| 82.11 | samtools | Calculate depth and coverage |
| 82.12 | GATK HaplotypeCaller | Call germline heterozygous SNVs and INDELs (only for bulk DNA) |
| 85.1 | SCcaller | Run SCcaller on all candidate mutations |
| 85.2 | n.a | Filter out low-quality and germline calls and keep only somatic mutations |
| 85.3 | bedtools | Calculate sensitivity |
| 85.4 | samtools | Calculate coverage of both bulk and single cell |
| 85.5 | n.a | Estimate SNV and INDEL burdens per single cell |