Methylation

Whole genome bisulfite sequencing is supported using the bismark2 pipeline. It can be turned on by setting analysis to wgbs-seq.

Supported Kits

Name directional? TrimR1_5’ TrimR1_3’ TrimR2_5’ TrimR2_3’
accelngs yes 0 nt 19 nt 19 nt 0
nebemseq yes 0 nt 0 0 0
truseq no 8 nt 8 8 8

Vendor links:

It is possible to specify the trim_galore trimming parameters and bismark directionality parameter (default: directional) in the resources section of the bcbio config file. The following configs for the truseq kit will give the same results:

details:
  - analysis: wgbs-seq
    genome_build: hg38
    algorithm:
      aligner: bismark
      kit: truseq
details:
  - analysis: wgbs-seq
    genome_build: hg38
    algorithm:
      aligner: bismark
    resources:
      trim_galore:
        options: ["--clip_r1 8", "--clip_r2 8", "--three_prime_clip_r1 8", "--three_prime_clip_r2 8"]
      bismark:
        options: ["--non_directional"]

Parameters

  • aligner: bismark
  • kit: accelngs, nebemseq, truseq

Benchmarking

There is an extensive discussion on Bismark and trim_galore performance, Bismark github. We ran a test with NA12878 nebemseq data, 125 mln reads (72.5mln read pairs). We tested performance of bismark/bcbio using --parallel (bismark workers) and -p (bowtie threads) bismark settings. We measured the performance only of the alignment step using bcbio-nextgen-commands log timecodes. 16/2/100G RAM was an optimal parameters set, with other having 5X-10X longer runtimes. When running a cohort of samples ~50% passed with 16/2/100G, some processed broken bam files, re-running with 8/2/100G or 4/2/100G solved the issue. For Lambda Phage genome we re-used trimming step results and 4/2/30G settings.

bcbio.yaml:

details:
- algorithm:
    aligner: bismark
    kit: nebemseq
  analysis: wgbs-seq
  description: NA12878_1
  files:
  - /path/to/NA12878_1.fq.gz
  - /path/to/NA12878_2fq.gz
  genome_build: hg38
  metadata:
    batch: NA12878_1-batch
    phenotype: tumor
fc_name: bcbio
resources:
  bismark:
    bismark_threads: 1
    bowtie_threads: 8
upload:
  dir: ../final

Tests:

test_N bismark_threads bowtie_threads time bcbio -n RAM
01 1 2 14h 42min 16 50G
02 1 4 >3.5 days 16 50G
03 1 8 1d 21h 16 50G
04 1 16 1d 15h 32 50G
05 1 32 >3day 14h 64 100G
06 2 2 2days 4h 16 50G
07 4 2 13h 16 50G
08 8 2 3h 34 min 16 50G
09 16 2 2h 42 min 32 100G
10 32 2 7h 40 min 64 250G
11 2 4 1d 23h 16 50G
12 2 8 11h 30 min 16 50G
13 2 16 11h 45 min 32 50G
14 2 32 >3days 14h 64 100G
15 4 2 4h 4 min 16 50G
16 4 4 15 h 16 50G
17 4 8 6 h 32 50G
18 4 16 15h 64 100G
19 4 32 1d 2h 128 200G
20 24 2 8h 48 192G