Question: chip-seq analysis (bowtie2, MACS2)
0
gravatar for marina-orlova
3.4 years ago by
Russian Federation
marina-orlova60 wrote:

Could you please explain where I made a mistake analyzing ChiP-seq data.

I took fastq file from modencode (set 2639):

http://data.modencode.org/cgi-bin/findFiles.cgi?download=2638,2639

then mapped it to dm3 genome (bowtie2). Then I took .sam file from its output and called peaks (MACS2). 

Final .bed file (with narrow peaks) is completely different from gff3 file from modencode:

Number of peaks differs about 2 times.

alignment macs2 fastq bowtie2 • 3.7k views
ADD COMMENTlink modified 3.4 years ago by tangming20052.2k • written 3.4 years ago by marina-orlova60
2
gravatar for Sukhdeep Singh
3.4 years ago by
Sukhdeep Singh9.1k
Netherlands
Sukhdeep Singh9.1k wrote:

It really depends on , what tools for mapping and peak calling were used along with the parameters. Slight change can cause differences, make sure the peakset you downloaded, has been analysed the same way. 

ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia

ADD COMMENTlink written 3.4 years ago by Sukhdeep Singh9.1k

So, if I have only raw data from modencode, should I use their guidelines to analyze it? I know my question seems stupid, sorry for that, I'm a newbie in bioinformatics.

ADD REPLYlink written 3.4 years ago by marina-orlova60

Its not stupid, you can use any method for analysing, if you know what you are doing. But using different methods to get the exactly same result is difficult. Mapping might be still fine (using different mapping algorithms) but peak callers can generate huge variability, depending on parameters and use of controls.
 

ADD REPLYlink written 3.4 years ago by Sukhdeep Singh9.1k

Ok, I understood you. To be clear: my goal is to define which genes are regulated by the TF. I'm trying to determine binding sites for TF. But if results of peak calling are completely different I suppose that the final results of analysis will differ. Am I right or not?

And could you please advise how to determine which parameters I should use in MACS2 (mfold, q-value, p-valus etc.) and in bowtie2 (alignment options, inpu options etc.)? Maybe any manual exists or learning videos. I tried using only default parameters earlier.

ADD REPLYlink written 3.4 years ago by marina-orlova60
2
gravatar for tangming2005
3.4 years ago by
tangming20052.2k
Houston/MD Anderson Cancer Center
tangming20052.2k wrote:

The results can be very different with different analyzing methods. No surprise at all.

ADD COMMENTlink written 3.4 years ago by tangming20052.2k
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 952 users visited in the last hour