Tag Archives: bismark

TrimGalore/FastQC/MultiQC – TrimGalore! RRBS Geoduck BS-seq FASTQ data


20180516 – UPDATE!!

THIS WAS RUN WITH THE INCORRECT SETTING IN TRIMGALORE! --non-directional

WILL RE-RUN


Steven requested that I trim the Geoduck RRBS libraries that we have, in preparation to run them through Bismark.

These libraries were originally created by Hollie Putnam using the TruSeq DNA Methylation Kit (Illumina):

All analysis is documented in a Jupyter Notebook; see link below.

Overview of process:

  1. Copy EPI* FastQ files from owl/P_generosa to roadrunner.

  2. Confirm data integrity via MD5 checksums.

  3. Run TrimGalore! with --paired, --rrbs, and --non-directional settings.

  4. Run FastQC and MultiQC on trimmed files.

  5. Copy all data to owl (see Results below for link).

  6. Confirm data integrity via MD5 checksums.

Jupyter Notebook:


Results:
TrimGalore! output folder:
FastQC output folder:
MultiQC output folder:
MultiQC report (HTML):
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BS-seq Mapping – Olympia oyster bisulfite sequencing: TrimGalore > FastQC > Bismark

Steven asked me to evaluate our methylation sequencing data sets for Olympia oyster.

According to our Olympia oyster genome wiki, we have the following two sets of BS-seq data:

All computing was conducted on our Apple Xserve: emu.

All steps were documented in this Jupyter Notebook (GitHub): 20180503_emu_oly_methylation_mapping.ipynb

NOTE: The Jupyter Notebook linked above is very large in size. As such it will not render on GitHub. It will need to be downloaded to a computer that can run Jupyter Notebooks and viewed that way.

Here’s a brief overview of what was done.

Samples were trimmed with TrimGalore and then evaluated with FastQC. MultiQC was used to generate a nice visual summary report of all samples.

The Olympia oyster genome assembly, pbjelly_sjw_01, was used as the reference genome and was prepared for use in Bismark:


/home/shared/Bismark-0.19.1/bismark_genome_preparation 
--path_to_bowtie /home/shared/bowtie2-2.3.4.1-linux-x86_64/ 
--verbose /home/sam/data/oly_methylseq/oly_genome/ 
2> 20180507_bismark_genome_prep.err

Bismark was run on trimmed samples with the following command:


/home/shared/Bismark-0.19.1/bismark 
--path_to_bowtie /home/shared/bowtie2-2.3.4.1-linux-x86_64/ 
--genome /home/sam/data/oly_methylseq/oly_genome/ 
-u 1000000 
-p 16 
--non_directional 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/1_ATCACG_L001_R1_001_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/2_CGATGT_L001_R1_001_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/3_TTAGGC_L001_R1_001_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/4_TGACCA_L001_R1_001_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/5_ACAGTG_L001_R1_001_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/6_GCCAAT_L001_R1_001_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/7_CAGATC_L001_R1_001_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/8_ACTTGA_L001_R1_001_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_10_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_11_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_12_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_13_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_14_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_15_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_16_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_17_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_18_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_1_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_2_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_3_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_4_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_5_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_6_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_7_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_8_s456_trimmed.fq.gz 
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_9_s456_trimmed.fq.gz 
2> 20180507_bismark_02.err

Results:

TrimGalore output folder:

FastQC output folder:

MultiQC output folder:

MultiQC Report (HTML):

Bismark genome folder: 20180503_oly_genome_pbjelly_sjw_01_bismark/

Bismark output folder:


Whole genome BS-seq (2015)

Prep overview
  • Library prep: Roberts Lab
  • Sequencing: Genewiz
Bismark Report Mapping Percentage
1_ATCACG_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 40.3%
2_CGATGT_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 39.9%
3_TTAGGC_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 40.2%
4_TGACCA_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 40.4%
5_ACAGTG_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 39.9%
6_GCCAAT_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 39.6%
7_CAGATC_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 39.9%
8_ACTTGA_L001_R1_001_trimmed_bismark_bt2_SE_report.txt 39.7%

MBD BS-seq (2015)

Prep overview
  • MBD: Roberts Lab
  • Library prep: ZymoResearch
  • Sequencing: ZymoResearch
Bismark Report Mapping Percentage
zr1394_1_s456_trimmed_bismark_bt2_SE_report.txt 33.0%
zr1394_2_s456_trimmed_bismark_bt2_SE_report.txt 34.1%
zr1394_3_s456_trimmed_bismark_bt2_SE_report.txt 32.5%
zr1394_4_s456_trimmed_bismark_bt2_SE_report.txt 32.8%
zr1394_5_s456_trimmed_bismark_bt2_SE_report.txt 35.2%
zr1394_6_s456_trimmed_bismark_bt2_SE_report.txt 35.5%
zr1394_7_s456_trimmed_bismark_bt2_SE_report.txt 32.8%
zr1394_8_s456_trimmed_bismark_bt2_SE_report.txt 33.0%
zr1394_9_s456_trimmed_bismark_bt2_SE_report.txt 34.7%
zr1394_10_s456_trimmed_bismark_bt2_SE_report.txt 34.9%
zr1394_11_s456_trimmed_bismark_bt2_SE_report.txt 30.5%
zr1394_12_s456_trimmed_bismark_bt2_SE_report.txt 35.8%
zr1394_13_s456_trimmed_bismark_bt2_SE_report.txt 32.5%
zr1394_14_s456_trimmed_bismark_bt2_SE_report.txt 30.8%
zr1394_15_s456_trimmed_bismark_bt2_SE_report.txt 31.3%
zr1394_16_s456_trimmed_bismark_bt2_SE_report.txt 30.7%
zr1394_17_s456_trimmed_bismark_bt2_SE_report.txt 32.4%
zr1394_18_s456_trimmed_bismark_bt2_SE_report.txt 34.9%
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