---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2018-05-08 17:40:06+00:00
layout: post
slug: bs-seq-mapping-olympia-oyster-bisulfite-sequencing-trimgalore-fastqc-bismark
title: 'BS-seq Mapping - Olympia oyster bisulfite sequencing: TrimGalore > FastQC
  > Bismark'
categories:
  - 2018
  - BS-seq Libraries for Sequencing at Genewiz
  - MBD Enrichment for Sequencing at ZymoResearch
tags:
  - bismark
  - BS-seq
  - FASTQC
  - jupyter notebook
  - multiqc
  - olympia oyster
  - Ostrea lurida
  - TrimGalore!
  - trimming
---

[Steven asked me to evaluate our methylation sequencing data sets for Olympia oyster](https://github.com/RobertsLab/resources/issues/225).

According to our [Olympia oyster genome wiki](https://github.com/RobertsLab/project-olympia.oyster-genomic/wiki), we have the following two sets of BS-seq data:





  * [Whole genome BS-seq (2015)](https://github.com/RobertsLab/project-olympia.oyster-genomic/wiki/Whole-genome-BSseq-December-2015)



  * [MBD BS-seq (2015)](https://github.com/RobertsLab/project-olympia.oyster-genomic/wiki/MBD-BSseq-December-2015)






All computing was conducted on our Apple Xserve: `emu`.

All steps were documented in this Jupyter Notebook (GitHub): [20180503_emu_oly_methylation_mapping.ipynb](https://github.com/sr320/LabDocs/blob/master/jupyter_nbs/sam/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](https://github.com/RobertsLab/project-olympia.oyster-genomic/wiki/Genome-Assemblies), was used as the reference genome and was prepared for use in Bismark:


<pre><code>
/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
</code></pre>



Bismark was run on trimmed samples with the following command:



<pre><code>
/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
</code></pre>





#### Results:



TrimGalore output folder:





  * [20180503_oly_methylseq_trimgalore](https://owl.fish.washington.edu/Athaliana/20180503_oly_methylseq_trimgalore/)



FastQC output folder:



  * [20180503_oly_methylseq_trimgalore/20180503_trim_fastqc/](https://owl.fish.washington.edu/Athaliana/20180503_oly_methylseq_trimgalore/20180503_trim_fastqc/)



MultiQC output folder:



  * [20180503_oly_methylseq_trimgalore/20180503_trim_fastqc/multiqc_data/](https://owl.fish.washington.edu/Athaliana/20180503_oly_methylseq_trimgalore/20180503_trim_fastqc/multiqc_data/)



MultiQC Report (HTML):



  * [20180503_oly_methylseq_trimgalore/20180503_trim_fastqc/multiqc_data/multiqc_report.html](https://owl.fish.washington.edu/Athaliana/20180503_oly_methylseq_trimgalore/20180503_trim_fastqc/multiqc_data/multiqc_report.html)



Bismark genome folder: [20180503_oly_genome_pbjelly_sjw_01_bismark/](https://owl.fish.washington.edu/Athaliana/20180503_oly_genome_pbjelly_sjw_01_bismark/)

Bismark output folder:





  * [20180507_oly_methylseq_bismark](https://owl.fish.washington.edu/Athaliana/20180507_oly_methylseq_bismark/)





* * *





#### [Whole genome BS-seq (2015)](https://github.com/RobertsLab/project-olympia.oyster-genomic/wiki/Whole-genome-BSseq-December-2015)





##### Prep overview







  * Library prep: Roberts Lab


  * Sequencing: Genewiz



<table >

<tr >
  Bismark Report
  Mapping Percentage
</tr>

<tbody >
<tr >

<td >1_ATCACG_L001_R1_001_trimmed_bismark_bt2_SE_report.txt
</td>

<td >40.3%
</td>
</tr>
<tr >

<td >2_CGATGT_L001_R1_001_trimmed_bismark_bt2_SE_report.txt
</td>

<td >39.9%
</td>
</tr>
<tr >

<td >3_TTAGGC_L001_R1_001_trimmed_bismark_bt2_SE_report.txt
</td>

<td >40.2%
</td>
</tr>
<tr >

<td >4_TGACCA_L001_R1_001_trimmed_bismark_bt2_SE_report.txt
</td>

<td >40.4%
</td>
</tr>
<tr >

<td >5_ACAGTG_L001_R1_001_trimmed_bismark_bt2_SE_report.txt
</td>

<td >39.9%
</td>
</tr>
<tr >

<td >6_GCCAAT_L001_R1_001_trimmed_bismark_bt2_SE_report.txt
</td>

<td >39.6%
</td>
</tr>
<tr >

<td >7_CAGATC_L001_R1_001_trimmed_bismark_bt2_SE_report.txt
</td>

<td >39.9%
</td>
</tr>
<tr >

<td >8_ACTTGA_L001_R1_001_trimmed_bismark_bt2_SE_report.txt
</td>

<td >39.7%
</td>
</tr>
</tbody>
</table>



* * *





#### [MBD BS-seq (2015) ](https://github.com/RobertsLab/project-olympia.oyster-genomic/wiki/MBD-BSseq-December-2015)





##### Prep overview







  * MBD: Roberts Lab


  * Library prep: ZymoResearch


  * Sequencing: ZymoResearch



<table >

<tr >
  Bismark Report
  Mapping Percentage
</tr>

<tbody >
<tr >

<td >zr1394_1_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >33.0%
</td>
</tr>
<tr >

<td >zr1394_2_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >34.1%
</td>
</tr>
<tr >

<td >zr1394_3_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >32.5%
</td>
</tr>
<tr >

<td >zr1394_4_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >32.8%
</td>
</tr>
<tr >

<td >zr1394_5_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >35.2%
</td>
</tr>
<tr >

<td >zr1394_6_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >35.5%
</td>
</tr>
<tr >

<td >zr1394_7_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >32.8%
</td>
</tr>
<tr >

<td >zr1394_8_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >33.0%
</td>
</tr>
<tr >

<td >zr1394_9_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >34.7%
</td>
</tr>
<tr >

<td >zr1394_10_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >34.9%
</td>
</tr>
<tr >

<td >zr1394_11_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >30.5%
</td>
</tr>
<tr >

<td >zr1394_12_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >35.8%
</td>
</tr>
<tr >

<td >zr1394_13_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >32.5%
</td>
</tr>
<tr >

<td >zr1394_14_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >30.8%
</td>
</tr>
<tr >

<td >zr1394_15_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >31.3%
</td>
</tr>
<tr >

<td >zr1394_16_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >30.7%
</td>
</tr>
<tr >

<td >zr1394_17_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >32.4%
</td>
</tr>
<tr >

<td >zr1394_18_s456_trimmed_bismark_bt2_SE_report.txt
</td>

<td >34.9%
</td>
</tr>
</tbody>
</table>
