Changes between Version 5 and Version 6 of SOPs/RRBS


Ignore:
Timestamp:
05/20/22 09:14:51 (3 years ago)
Author:
twhitfie
Comment:

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  • SOPs/RRBS

    v5 v6  
    66
    77=== Step by step analysis ===
     8
     9* **Genome indexing**
     10    * Bismark relies on indexed reference genomes (for short read mapping using [https://genomebiology.biomedcentral.com/articles/10.1186/gb-2009-10-3-r25 bowtie] or [https://www.nature.com/articles/nmeth.1923 bowtie2]) that have been ''in silico'' bisulfite treated to create C-to-T and G-to-A versions of the reference.
     11    * Several reference genomes (e.g. human, mouse, fly) are already available in /nfs/genomes, so this indexing step will only be necessary if you are not using one of these assemblies.
     12    * For a custom reference genome, the Bismark indexing can be done with:
     13
     14{{{
     15bsub bismark_genome_preparation --bowtie2 /path/to/organism/reference/assembly/fasta
     16}}}
     17
     18   * The above command will write the indices needed by bismark to /path/to/organism/reference/assembly/fasta/Bisulfite_Genome.  When running bismark, however, point to one directory level above this (i.e./path/to/organism/reference/assembly/fasta).  In the example below, a reference on /nfs/genomes is indicated.
    819
    920  * **Quality control**
    … …  
    1829  * **Alignment and methylation calls**
    1930    * Bismark can (since version 0.6.beta1) use [https://www.nature.com/articles/nmeth.1923 bowtie2] to map short reads to reference genome(s).
    20     * 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.
     31    * In the command below, bismark will expect to find C-to-T and 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.
    2132    * [https://www.bioinformatics.babraham.ac.uk/projects/bismark/ Bismark]  produces BAM file(s) of aligned reads and methylation calls.
    2233