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Thursday, January 23, 2014

MeDIP-seq and histone modification ChIP-seq analysis

I was reading a cell paper:

Dnmt3L Antagonizes DNA Methylation at Bivalent Promoters and Favors DNA Methylation at Gene Bodies in ESCs

http://www.sciencedirect.com.lp.hscl.ufl.edu/science/article/pii/S0092867413010878
I want to re-analyze the MeDIP-seq data and the histone modification ChIP-seq data.

I downloaded the MeDIP-seq data here: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44644
and the H3k4me3 and H3k27me3 ChIP-seq data here: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15519

The paper claimed that Dnmt3L maintains DNA hypomethylation at bivalent promoters ( with both H3k4me3 and H3k27me3 marks).  The authors used a Dnmt3L deficient mouse ES cell line and did MeDIP-seq on it. They found that many bivalent promoters gain DNA methylation in Dnmt3L negative cells. In contrast, DNA methylation loss mainly occurs in gene body.

I will work on the histone modification ChIP-seq data first.
I took a look at many different H3k4me3 and H3k27me3 ChIP-seq data sets in mES cells. H3k4me3 ChIP-seq data quality is usually very good with very specific and sharp peaks after MACS peak calling. However, H3k27me3 data are not that great. I did regular H3k4me3 ChIP, the antibody is very good and the enrichment of input is usually above 10% in the active loci I tested.

Similar to H3k36me3, an active mark in gene body, H3k27me3 (repressive) mark usually form broader but less-enriched regions. I played around with the MACS argument setting.

I will skip the process of transforming sra format data to fastq and mapping them to the reference genome. I have several posts for that already:
http://crazyhottommy.blogspot.com/2013/09/the-backtick-in-linux-command.html
bowtie for ChIp-seq http://crazyhottommy.blogspot.com/2013/05/sra-file-and-my-first-bowtie-run-on-uf.html
Tophat for RNA-seq http://crazyhottommy.blogspot.com/2013/10/rna-seq-analysis-samtools-sort-and.html

For H3k4me3 data, I only downloaded one, and called peaks:
[mtang@dev1 H3k4me3]$ macs -t SRR038984.sam -n initial_test -g mm 
INFO  @ Thu, 23 Jan 2014 13:37:47: 
# ARGUMENTS LIST:
# name = initial_test
# format = AUTO
# ChIP-seq file = SRR038984.sam
# control file = None
# effective genome size = 1.87e+09
# band width = 300
# model fold = 10,30
# pvalue cutoff = 1.00e-05
# Large dataset will be scaled towards smaller dataset.
# Range for calculating regional lambda is: 10000 bps

INFO  @ Thu, 23 Jan 2014 13:37:47: #1 read tag files... 
INFO  @ Thu, 23 Jan 2014 13:37:47: #1 read treatment tags... 
INFO  @ Thu, 23 Jan 2014 13:37:47: Detected format is: SAM 
INFO  @ Thu, 23 Jan 2014 13:37:56:  1000000 
INFO  @ Thu, 23 Jan 2014 13:38:05:  2000000 
INFO  @ Thu, 23 Jan 2014 13:38:13:  3000000 
INFO  @ Thu, 23 Jan 2014 13:38:21:  4000000 
INFO  @ Thu, 23 Jan 2014 13:38:29:  5000000 
INFO  @ Thu, 23 Jan 2014 13:38:38:  6000000 
INFO  @ Thu, 23 Jan 2014 13:38:46:  7000000 
WARNING @ Thu, 23 Jan 2014 13:38:56: NO records for chromosome chr16_random, minus strand! 
INFO  @ Thu, 23 Jan 2014 13:38:56: #1 tag size is determined as 36 bps 
INFO  @ Thu, 23 Jan 2014 13:38:56: #1 tag size = 36 
INFO  @ Thu, 23 Jan 2014 13:38:56: #1  total tags in treatment: 5000614 
INFO  @ Thu, 23 Jan 2014 13:38:56: #1 user defined the maximum tags... 
INFO  @ Thu, 23 Jan 2014 13:38:56: #1 filter out redundant tags at the same location and the same strand by allowing at most 1 tag(s) 
INFO  @ Thu, 23 Jan 2014 13:38:57: #1  tags after filtering in treatment: 3982997 
INFO  @ Thu, 23 Jan 2014 13:38:57: #1  Redundant rate of treatment: 0.20 
INFO  @ Thu, 23 Jan 2014 13:38:57: #1 finished! 
INFO  @ Thu, 23 Jan 2014 13:38:57: #2 Build Peak Model... 
INFO  @ Thu, 23 Jan 2014 13:39:15: #2 number of paired peaks: 22337 
INFO  @ Thu, 23 Jan 2014 13:39:16: #2 finished! 
INFO  @ Thu, 23 Jan 2014 13:39:16: #2 predicted fragment length is 164 bps 
INFO  @ Thu, 23 Jan 2014 13:39:16: #2.2 Generate R script for model : initial_test_model.r 
INFO  @ Thu, 23 Jan 2014 13:39:16: #3 Call peaks... 
INFO  @ Thu, 23 Jan 2014 13:39:16: #3 shift treatment data 
INFO  @ Thu, 23 Jan 2014 13:39:17: #3 merge +/- strand of treatment data 
INFO  @ Thu, 23 Jan 2014 13:39:18: #3 call peak candidates 
INFO  @ Thu, 23 Jan 2014 13:40:14: #3 use self to calculate local lambda and  filter peak candidates... 
INFO  @ Thu, 23 Jan 2014 13:40:27: #3 Finally, 32919 peaks are called! 
INFO  @ Thu, 23 Jan 2014 13:40:27: #4 Write output xls file... initial_test_peaks.xls 
INFO  @ Thu, 23 Jan 2014 13:40:27: #4 Write peak bed file... initial_test_peaks.bed 
INFO  @ Thu, 23 Jan 2014 13:40:27: #4 Write summits bed file... initial_test_summits.bed 
INFO  @ Thu, 23 Jan 2014 13:40:27: #5 Done! Check the output files! 

MACS successfully built the model and identified 32919 peaks.

For H3k27me3 data, there are two replicates, I merged them with samtools merge first

[mtang@dev1 H3k27me3]$ samtools merge merged_H3k27me3.bam SRR038975.bam SRR038976.bam 

then at HPC@UFL:

[mtang@dev1 H3k27me3]$ macs -t merged_H3k27me3.bam  -n initial_test -g mm 

it gave me only 4941 peaks.

[mtang@dev1 H3k27me3]$ wc -l merged_H3k27me3_peaks.bed 
4941 merged_H3k27me3_peaks.bed

I changed the arguments:
[mtang@dev1 H3k27me3]$ macs -t merge.bam -n no_model  -g mm --nomodel --shiftsize 73 --pvalue 1e-3 

note that I specify no peaking model building, shift size 73 (  fragment size 73x2=146 bp, a nuclesome size), and a less stringent pvalue 1e-3 ( the default is 1e-5)

This time I got 24522 peaks.

[mtang@dev1 H3k27me3]$ wc -l no_model_peaks.bed 
24522 no_model_peaks.bed

In my next post, I will use those two peak bed files generated by MACS to find the bivalent promoters.
=================================================================

I will switch gear and analyze the MeDIP-seq data. MeDIP-seq data are essentially similar to ChIP-seq data. Although there are specific software to analyze it, MACS  works well with this kind of data.

see a list of programs here http://seqanswers.com/wiki/Software/list

I have  MeDIP_shGFP.sam and MeDIP-shDnmt3L.sam files ready. 

To visualize the MeDIP data in IGV, I have the bedgraph files generated by MACS respectively. I also want to have a bedgraph file that with MeDIP-shGFP signal subtracted from MeDIP-shDnmt3L signal.

MACS2 has a bdgcpm program for that purpose

macs2 bdgcmp -t shDnmt3L_treat_afterfiting_all.bdg -c shGFP_treat_afterfiting_all.bdg -o subtracted.bdg -m subtract

Now, I have almost every file for visualization. Just to reproduce fig4E in the paper:
The first track is the MeDIP-seq signal for shGFP; the second is the MeDIP-seq signal for shDnmt3L; the third is the shDnmt3L signal subtracted with shGFP signal; the fourth track is the H3k4me3 ChIP-seq signal; the fifth is the H3k4me3 peaks called by MACS; the sixth is the H3k27me3 ChIP-seq signal; the last is the H3k27me3 peaks called by MACS.















The next question is to find the differentially changed peaks after ablating Dnmt3L. I have a post discussing this topic before http://crazyhottommy.blogspot.com/2013/10/compare-chip-seq-data-for-different.html

MACS2 has some new implements for this kind of purpose, but it requires a IgG or Input control file for each condition. https://github.com/taoliu/MACS/wiki/Call-differential-binding-events

I only have two bam files, so I will use http://code.google.com/p/diffreps/
diffReps needs  bed files to be the input. I converted the bam files to bed files with bedtools first:

[mtang@dev1 MeDIP-seq]$ bamToBed -i SRR764931_shGFP.bam > SRR764931_shGFP.bed
[mtang@dev1 MeDIP-seq]$ bamToBed -i SRR764932_shDnmt3L.bam > SRR764932_shDnmt3L.bed

since I do not have biological replicates, I used G-test 

[mtang@dev1 MeDIP-seq]$ diffReps.pl -tr SRR764932_shDnmt3L.bed -co SRR764931_shGFP.bed -gn mm9 -re diff.nb.txt -me gt

It generates three files:
diff.nb.txt
diff.nb.txt.annotated
diff.nb.txt.hotspot

for more information of these three files, see the diffReps link above.









3 comments:

  1. some other people also used diffReps for MeDIP data :)
    http://ethanomics.wordpress.com/2014/02/19/medip-seq-data-analysis-diffreps/

    ReplyDelete
  2. just a lame question, but what files correspond to the fourth and sixth tracks? thank you!

    ReplyDelete
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