BSMAP-methatio-methykit workflow

Assumptions- as with most worflows in development - a working directory is used.

In [49]: 
#SR edits - adding direct link to ipynb viewer and raw files
!date 

Thu Feb 20 19:23:24 PST 2014
In [43]: 
filename='BSMAP2MK_workflow'

ipynb viewer format:

In [44]: 
!echo 'http://nbviewer.ipython.org/github/sr320/ipython_nb/blob/master/''{filename}''.ipynb'

http://nbviewer.ipython.org/github/sr320/ipython_nb/blob/master/BSMAP2MK_workflow.ipynb

ipynb file:

In [48]: 
!echo 'http://nbviewer.ipython.org/github/sr320/ipython_nb/blob/master/''{filename}''.ipynb'

http://nbviewer.ipython.org/github/sr320/ipython_nb/blob/master/BSMAP2MK_workflow.ipynb

Setting Variables

In [1]: 
#file ID
fid="CgF_nov"

#TIMESTAMP
date=!date +%m%d_%H%M
#working directory (parent)
wd="/Volumes/web/Mollusk/bs_larvae_exp/"

#where is bsmap
bsmap="/Users/Shared/Apps/bsmap-2.73/"

#fastq files location R1 location
R1="/Volumes/web/trilobite/Crassostrea_gigas_HTSdata/batterbox/FCC39EM/Sample_BS_CgF/filtered_BS_CgF_TTAGGC_L004_R1.fastq.gz"

#fastq files location R2 location
#comment out if SE
#R2="/Volumes/web/trilobite/Crassostrea_gigas_HTSdata/batterbox/FCC39EM/Sample_BS_CgF/filtered_BS_CgF_TTAGGC_L004_R1.fastq.gz"


#genome file 
genome="/Volumes/web/whale/ensembl/ftp.ensemblgenomes.org/pub/release-21/metazoa/fasta/crassostrea_gigas/dna/Crassostrea_gigas.GCA_000297895.1.21.dna_sm.genome.fa"
In [2]: 
cd {wd}

/Volumes/web/Mollusk/bs_larvae_exp
In [6]: 
mkdir {fid}_{date}
In [7]: 
cd {fid}_{date}

/Volumes/web/Mollusk/bs_larvae_exp/CgF_nov
In [8]: 
#option - number of processes 
!{bsmap}bsmap -a {R1} -d {genome} -o bsmap_out.sam -p 1


BSMAP v2.73
Start at:  Wed Feb 12 16:25:40 2014

Input reference file: /Volumes/web/whale/ensembl/ftp.ensemblgenomes.org/pub/release-21/metazoa/fasta/crassostrea_gigas/dna/Crassostrea_gigas.GCA_000297895.1.21.dna_sm.genome.fa 	(format: FASTA)
Load in 7658 db seqs, total size 557717710 bp. 9 secs passed
total_kmers: 43046721
Create seed table. 25 secs passed
max number of mismatches: read_length * 8% 	max gap size: 0
kmer cut-off ratio:5e-07
max multi-hits: 100	max Ns: 5	seed size: 16	index interval: 4
quality cutoff: 0	base quality char: '!'
min fragment size:28	max fragemt size:500
start from read #1	end at read #4294967295
additional alignment: T in reads => C in reference
mapping strand: ++,-+
Single-end alignment(1 threads)
Input read file: /Volumes/web/trilobite/Crassostrea_gigas_HTSdata/batterbox/FCC39EM/Sample_BS_CgF/filtered_BS_CgF_TTAGGC_L004_R1.fastq.gz 	(format: gzipped FASTQ)
Output file: bsmap_out.sam	 (format: SAM)
Thread #0: 	50000 reads finished. 29 secs passed
Thread #0: 	100000 reads finished. 34 secs passed
Thread #0: 	150000 reads finished. 39 secs passed
Thread #0: 	200000 reads finished. 43 secs passed
Thread #0: 	250000 reads finished. 48 secs passed
Thread #0: 	300000 reads finished. 53 secs passed
Thread #0: 	345868 reads finished. 57 secs passed
Total number of aligned reads: 218893 (63%)
Done.
Finished at Wed Feb 12 16:26:38 2014
Total time consumed:  58 secs
In [9]: 
!python {bsmap}methratio.py -d {genome} -u -z -g -o methratio_out.txt -s {bsmap}samtools bsmap_out.sam

@ Wed Feb 12 16:26:38 2014: reading reference /Volumes/web/whale/ensembl/ftp.ensemblgenomes.org/pub/release-21/metazoa/fasta/crassostrea_gigas/dna/Crassostrea_gigas.GCA_000297895.1.21.dna_sm.genome.fa ...
@ Wed Feb 12 16:27:11 2014: reading bsmap_out.sam ...
[samopen] SAM header is present: 7658 sequences.
@ Wed Feb 12 16:27:19 2014: combining CpG methylation from both strands ...
@ Wed Feb 12 16:27:48 2014: writing methratio_out.txt ...
@ Wed Feb 12 16:29:45 2014: done.
total 168921 valid mappings, 1626861 covered cytosines, average coverage: 1.11 fold.

Filter the methratio output file so that I'm only obtaining the context '_CG' and loci with at least 5x coverage

In []: 
#command for only obtaining the context '__CG_'
!grep "[A-Z][A-Z]CG[A-Z]" <methratio_out.txt> methratio_out_CG.txt
In [15]: 
#obtaining a filtered file with at least 5x coverage
!awk '$8 >= 5' <methratio_out_CG.txt> methratio_out_CG5x.txt
In [16]: 
#Now I need to format my files to be read into the methylKit package in R
In [12]: 
!/Volumes/web/Mollusk/bs_larvae_exp/methratio.awk.sh methratio_out_CG5x.txt > methratio_out_CG_mkit.txt
In [13]: 
!head methratio_out_CG_mkit.txt

chr.Base	chr	base	strand	coverage	freqC	freqT
C12802.90	C12802	90	F	1	0.00	1.00
C12802.99	C12802	99	F	1	0.00	1.00
C12802.119	C12802	119	F	1	0.00	1.00
C12802.149	C12802	149	F	1	0.00	1.00
C12960.67	C12960	67	F	1	0.00	1.00
C12960.123	C12960	123	F	1	0.00	1.00
C13208.148	C13208	148	F	1	1.00	0.00
C13208.184	C13208	184	F	1	1.00	0.00
C13766.145	C13766	145	F	1	0.00	1.00
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