Workflow: Analysis of Oyster DNA Methylation Data

Posted in tumblelog

This has been reposted from our lab tumblelog

This workflow outlines analysis of data from mapping bisulfite treated DNA (BS-seq) to the oyster genome.

Initially sequencing reads, in this case from oyster sperm, are mapped to the genome using BS-MAP.

./bsmap -a /Volumes/betty/filtered_174gm_A_NoIndex_L006_R1.fastq.gz -b /Volumes/betty/filtered_174gm_A_NoIndex_L006_R2.fastq.gz -d /Volumes/betty/oyster_v9.fa -o /Volumes/betty/BiGO_Betty_plain.sam -p 4

output:

Total number of aligned reads:

pairs: 90102067 (53%)

single a: 17033002 (9.9%)

single b: 15975991 (9.3%)

http://bit.ly/14acMLn

Resulting SAM file then subjected to methratio python script

python methratio.py -d /Volumes/betty/oyster_v9.fa -u -z -g -o  /Volumes/betty/BiGO_betty_plain_methratio_v1.txt -s /Volumes/Bay3/Software/BSMAP/bsmap-2.73/samtools /Volumes/betty/BiGO_Betty_plain.sam

output:

total 167774088 valid mappings, 127192776 covered cytosines, average coverage: 13.23 fold.

http://bit.ly/14acOCZ

This file was uploaded to SQLShare using python script

python singleupload.py sr320@washington.edu c8APIKEYAPIKEYAPIKEY5c15c /Volumes/web/cnidarian/BiGO_betty_plain_methratio_v1.txt


directlink:
http://bit.ly/10CcIq5


Interested in is how many CpG loci is there information for?

The following query will get all lines where CG are in the 3rd and 4th position.

SELECT * FROM [sr320@washington.edu].[BiGO_betty_plain_methratio_v1.txt] betty where context like '__CG_' --_=single character wildcard



To count the number of instances in this query



How to convert methratio file to gff format

In this case will make GFF only of CpGs, where there is at least 10x coverage.

SELECT 
chr as seqname,  
'methratio' as source,  
'CpG' as feature, 
pos as start,
pos + 1 as [end],
ratio as score,  
strand,  
'.' as frame,  
'.' as attribute  

FROM [sr320@washington.edu].     
[BiGO_betty_plain_methratio_v1.txt] betty 
where 
context like '__CG_' --_=single character wildcard
and
CT_Count > 9`





http://bit.ly/10CcIqb





via Tidal Cycles – oystergen.es http://bit.ly/10CcFe9

via the Lab Tumblr: http://genefish.tumblr.com/post/53208753511