Changes between Version 3 and Version 4 of SOPs/RRBS

Timestamp:

05/19/22 17:14:06 (3 years ago)

Author:

twhitfie

Comment:

--

SOPs/RRBS

@@ -3 +3 @@
 === Background ===
 
-    * Reduced-representation bisulfite sequencing (RRBS) is an experimental [https://academic.oup.com/nar/article/33/18/5868/2401288 protocol] to measure and compare genomic methylation patterns.  The experiments use one or multiple restriction enzymes on genomic DNA to produce sequence specific fragments that are subsequently treated with bisulfite and sequenced. The workflow below illustrates how to use [https://academic.oup.com/bioinformatics/article/27/11/1571/216956 bismark] to analyze RRBS data on the resources at the Whitehead Institute. 
+    * Reduced-representation bisulfite sequencing (RRBS) is an experimental [https://academic.oup.com/nar/article/33/18/5868/2401288 protocol] to measure and compare genomic methylation patterns.  These experiments use one or multiple restriction enzymes on genomic DNA to produce sequence specific fragments that are subsequently treated with bisulfite and sequenced. The workflow below illustrates how to use [https://academic.oup.com/bioinformatics/article/27/11/1571/216956 bismark] to analyze RRBS data on the resources at the Whitehead Institute. 
 
 === Step by step analysis ===
 
-  * **QC**
-    * Use [https://www.bioinformatics.babraham.ac.uk/projects/trim_galore Trim Galore] or [http://barcwiki.wi.mit.edu/wiki/SOPs/qc_shortReads another] read trimmer to apply quality filters and remove adapters.
+  * **Quality control**
+    * Start by using [https://www.bioinformatics.babraham.ac.uk/projects/trim_galore Trim Galore] or [http://barcwiki.wi.mit.edu/wiki/SOPs/qc_shortReads another] read trimmer to apply quality filters and remove adapters.
     * See our [http://barcwiki.wi.mit.edu/wiki/SOPs/qc_shortReads QC and preprocessing guidelines] for details on running Trim Galore.
-    * A command for Trim Galore  paired end reads using gzipped fastq input can look like:
+    * A command for running Trim Galore on paired end reads from RRBS experiments can look like:
 
 {{{
@@ -17 +17 @@
 
   * **Quantification of methylation calls**
+    * Bismark can (since version 0.6.beta1) use [https://www.nature.com/articles/nmeth.1923 bowtie2] to map short reads to reference genome(s).
+    * In the command below, bismark will expect to find C-to-T and a G-to-A versions of the reference genome.  From the following the steps above, these will be located within /nfs/genomes/myOrganism/bowtie/ (for Institute-wide reference genomes) or /path/to/custom/genome/bowtie for other reference genomes.
     * [https://www.bioinformatics.babraham.ac.uk/projects/bismark/ Bismark]  produces BAM file(s) of aligned reads and methylation calls.
  
 {{{
-bsub /path/to/bismark/bismark --genome /nfs/genomes/mouse_mm10_dec_11_no_random/bowtie/ -1 trimmedReads_1.fq.gz -2 trimmedReads_2.fq.gz
+bsub bismark --genome /nfs/genomes/myOrganism/bowtie/ -1 trimmedReads_1.fq.gz -2 trimmedReads_2.fq.gz
 }}}
 
@@ -26 +28 @@
     * In the command above, bismark will expect to find C-to-T and a G-to-A versions of the reference genome.
 
-* **Extract methylation calls**
-    * After running bismark...
-    * For paired ended data...:
+* **Extract methylation percentages**
+    * After running bismark, the methylation percentages can be extracted from the BAM output using bismark_methylation_extractor.
+    * For paired ended data, use the "-p" flag:
 
 {{{
-bsub /path/to/bismark/bismark_methylation_extractor -p --gzip --bedGraph trimmedReads_bismark_bt2_pe.bam 
+bsub bismark_methylation_extractor -p --gzip --bedGraph trimmedReads_bismark_bt2_pe.bam 
 }}}
-    * Comment 1
-    * Comment 2
+    * The resulting file trimmedReads_bismark bt2_pe.bismark.cov.gz summarizes coverage in the following [https://github.com/FelixKrueger/Bismark/blob/master/Docs/README.md format]:
+ 
+{{{   
+<chromosome> <start position> <end position> <methylation percentage> <count methylated> <count unmethylated>
+}}}