Alignment¶
See the alignment hydra-genetics module documentation for more details on the softwares. Default hydra-genetics settings/resources are used if no configuration is specified.
Pipeline output files:¶
bam_dna/{sample}_{type}.bambam_dna/{sample}_{type}.bam.baibam_dna/{sample}_{type}.umi.bam(ctDNA only)bam_dna/{sample}_{type}.umi.bam.bai(ctDNA only)
Alignment (FFPE)¶
Alignment of fastq files into bam files is performed by bwa-mem v0.7.17 using the non-merged trimmed fastq files. This make it possible to speed up alignent by utlizing parallization and also make it possible to analyze qc for lanes separately. Bamfile are then directly sorted by samtools sort v1.15.
Alignment (ctDNA)¶
Alignment of ctDNA samples is performed using bwa-mem v0.7.17. To handle UMI (Unique Molecular Identifier) tags, the reads are processed to consensus reads to correct for PCR and sequencing errors. This is done using fgbio.
The UMI processing involves the following steps:
1. Copy UMI: Copies the UMI at the end of the BAM’s read name to the RX tag using fgbio CopyUmiFromReadName.
2. Group Reads: Group and sort reads based on UMI using fgbio GroupReadsByUmi.
3. Call Consensus: Call and filter consensus reads based on UMIs using fgbio CallDuplexConsensusReads followed by fgbio FilterConsensusReads.
Read groups¶
Bam file read groups are set according to sequencing information in the units.tsv file.
The @RG read tag is set using the following options defined in the hydra-genetics bwa rule:
-R '@RG\tID:{ID}\tSM:{SM}\tPL:{PL}\tPU:{PU}\tLB:{LB}' -v 1
where the individual read groups are defined below:
| RG tag | Value |
|---|---|
| ID | sample_type.lane.barcode |
| SM | sample_type |
| PL | platform |
| PU | flowcell.lane.barcode |
| LB | sample_type |
Configuration¶
Reference files
Software settings
| Options | Value | Description |
|---|---|---|
| sorting | samtools | use samtools to sort the bam files |
| sort_order | coordinate | use coordinate sorting |
| sort_extra | -@ 10 | use 10 threads for sorting |
Cluster resources
| Options | Value |
|---|---|
| mem_mb | 61440 |
| mem_per_cpu | 6144 |
| threads | 10 |
| time | "8:00:00" |
Bam splitting¶
The bam files are split into chromosome files for faster performance in downstream analysis. Split files are used by markduplicates and SNV/INDEL calling. Splitting is performed by samtools view v1.15.
Mark duplicates¶
Flagging duplicated reads are performed on individual chromosome bam files by picard MarkDuplicates v2.25.0.
Merging¶
Merging of deduplicated bam files belonging to the same sample are performed by samtools merge v1.15.
Configuration¶
Software settings
| Options | Value | Description |
|---|---|---|
| extra | -c | emit only one identical @RG headers |
| extra | -p | use the @PG ID of the first file |
Sorting¶
Merged bamfile are sorted by samtools sort v1.15.
Bam indexing¶
Bamfile indexing is performed by samtools index v1.15.