expected percentage of mapped reads in chip-seq experiment

andreiafonseca · 03-25-2011, 02:42 AM

Dear all,

I am starting to work with Chip-seq data to identify binding sites of transcription factors and we have just received a test run from the company in order to decide to go ahead with the sequencing. We are using Hiseq. For a test run we got 100,000 reads of each library. We have sequenced for each sample the correspondent input. For the samples 40-50% reads mapped to the human genome (allowing max 2 mismaches) whereas the input had a percentage of mapped reads ~90% . I was expecting to obtain a lower percentage of mapped reads in the input and not in the sample. I used bowtie to make the alignment. I would like to know if someone as had similar percentages of mapped reads in Chip-seq experiments.
Thanks

Andreia

kopi-o · 03-25-2011, 02:59 AM

This could depend on a lot of factors but 40-50% sounds low on the face of it. How does the quality score distribution of the ChIPped sequences look compared to the input? What read lengths are you using?

andreiafonseca · 03-25-2011, 03:12 AM

Thanks for answering! The read length is 49 bp. What do you mean by the quality score distibution? I have checked the average of the QS per position on the read and the quality score distribution is similar between the sample and the input. In the 5' end we have an average ~38 and at the 3'end ~34.

andreiafonseca · 03-25-2011, 03:34 AM

one more detail is single-end sequencing

kopi-o · 03-25-2011, 03:38 AM

I was thinking that maybe the quality scores would be lower at the 3' end for the sample, but that doesn't appear to be the case. I'm not sure what the explanation could be then, maybe something in the sample preparation?

andreiafonseca · 03-25-2011, 03:45 AM

it could happen that the enrichment was far from perfect, but why does the input has such a high proportion of mapped reads? shouldn't the input have a lower proportion because it is genomic DNA, so it has repeats, telomeric regions which will map to many locations?

kopi-o · 03-25-2011, 03:56 AM

Well, maybe ... but input DNA sequencing also does not give an unbiased representation of the genome; open-chromatin regions like TSS are overrepresented there too, see e g

http://www.plosone.org/article/info:...l.pone.0005241
http://www.biomedcentral.com/1471-2164/12/134

ttnguyen · 03-25-2011, 04:08 AM

Just wondering "mapped reads" are unique (I mean set -m 1 in Bowtie)? If so, probably 90% of mapped reads (only 49 bp in length) seems so high as ~50% of the human genome are masked by RepeatMasker?

BTW, It would be helpful to see the distribution of sequence quality by using fastQC:
http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/

andreiafonseca · 03-25-2011, 05:27 AM

thanks for your message. I took a bit because I was checking how many reads were uniquely mapped. In samples ~40-50% were uniquely mapped and in input ~80%. In attachment you can see the distribution of the QS, Lib1 is a sample and Lib2 is the corresponding input.
thanks for the help

ttnguyen · 03-25-2011, 05:44 AM

I could not find the attachment

. I think 40-50% in samples is normal, but 80% in input is quite high. Would it be worth marking PCR duplicate, or comparing your mapping rates with the public datasets (e.g. ENCODE - I think they had Input as well)?

andreiafonseca · 03-25-2011, 05:47 AM

sorry just noticed there was a problem in the attachment

andreiafonseca · 03-25-2011, 05:49 AM

this image is for the sample the previous one was for input

andreiafonseca · 03-25-2011, 05:50 AM

can you explain me what do you mean by marking PCR duplicate?

ttnguyen · 03-25-2011, 05:59 AM

This is very good QS I think as the average is high and the variation is low (even though at 3' end). Could you show your Bowtie command?

andreiafonseca · 03-25-2011, 06:03 AM

-f -a --best --strata -v 2 hg19 fasta

then I selected from these the unique alignments

can you tell me what do you mean by marking PCR duplicate?

ttnguyen · 03-25-2011, 06:06 AM

Quote:

Originally Posted by andreiafonseca

can you explain me what do you mean by marking PCR duplicate?

The DNA fragments maybe duplicated due to PCR amplification. It means you may have many reads mapped to the same position. Picard can help with removing duplicate.
http://picard.sourceforge.net/comman...overview.shtml

You can find more information about that in this forum as well.

ttnguyen · 03-25-2011, 06:14 AM

I think that is fine when you chose the -v alignment mode since the QS is good.

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03-25-2011, 02:42 AM	#1
andreiafonseca Junior Member Location: Portugal Join Date: Mar 2008 Posts: 9	expected percentage of mapped reads in chip-seq experiment Dear all, I am starting to work with Chip-seq data to identify binding sites of transcription factors and we have just received a test run from the company in order to decide to go ahead with the sequencing. We are using Hiseq. For a test run we got 100,000 reads of each library. We have sequenced for each sample the correspondent input. For the samples 40-50% reads mapped to the human genome (allowing max 2 mismaches) whereas the input had a percentage of mapped reads ~90% . I was expecting to obtain a lower percentage of mapped reads in the input and not in the sample. I used bowtie to make the alignment. I would like to know if someone as had similar percentages of mapped reads in Chip-seq experiments. Thanks Andreia

03-25-2011, 02:59 AM	#2
kopi-o Senior Member Location: Stockholm, Sweden Join Date: Feb 2008 Posts: 319	This could depend on a lot of factors but 40-50% sounds low on the face of it. How does the quality score distribution of the ChIPped sequences look compared to the input? What read lengths are you using?

03-25-2011, 03:12 AM	#3
andreiafonseca Junior Member Location: Portugal Join Date: Mar 2008 Posts: 9	Thanks for answering! The read length is 49 bp. What do you mean by the quality score distibution? I have checked the average of the QS per position on the read and the quality score distribution is similar between the sample and the input. In the 5' end we have an average ~38 and at the 3'end ~34.

03-25-2011, 03:34 AM	#4
andreiafonseca Junior Member Location: Portugal Join Date: Mar 2008 Posts: 9	one more detail is single-end sequencing

03-25-2011, 03:38 AM	#5
kopi-o Senior Member Location: Stockholm, Sweden Join Date: Feb 2008 Posts: 319	I was thinking that maybe the quality scores would be lower at the 3' end for the sample, but that doesn't appear to be the case. I'm not sure what the explanation could be then, maybe something in the sample preparation?

03-25-2011, 03:45 AM	#6
andreiafonseca Junior Member Location: Portugal Join Date: Mar 2008 Posts: 9	it could happen that the enrichment was far from perfect, but why does the input has such a high proportion of mapped reads? shouldn't the input have a lower proportion because it is genomic DNA, so it has repeats, telomeric regions which will map to many locations?

03-25-2011, 03:56 AM	#7
kopi-o Senior Member Location: Stockholm, Sweden Join Date: Feb 2008 Posts: 319	Well, maybe ... but input DNA sequencing also does not give an unbiased representation of the genome; open-chromatin regions like TSS are overrepresented there too, see e g http://www.plosone.org/article/info:...l.pone.0005241 http://www.biomedcentral.com/1471-2164/12/134

03-25-2011, 04:08 AM	#8
ttnguyen Member Location: Ireland Join Date: Mar 2010 Posts: 41	Just wondering "mapped reads" are unique (I mean set -m 1 in Bowtie)? If so, probably 90% of mapped reads (only 49 bp in length) seems so high as ~50% of the human genome are masked by RepeatMasker? BTW, It would be helpful to see the distribution of sequence quality by using fastQC: http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/

03-25-2011, 05:27 AM	#9
andreiafonseca Junior Member Location: Portugal Join Date: Mar 2008 Posts: 9	thanks for your message. I took a bit because I was checking how many reads were uniquely mapped. In samples ~40-50% were uniquely mapped and in input ~80%. In attachment you can see the distribution of the QS, Lib1 is a sample and Lib2 is the corresponding input. thanks for the help

03-25-2011, 05:44 AM	#10
ttnguyen Member Location: Ireland Join Date: Mar 2010 Posts: 41	I could not find the attachment . I think 40-50% in samples is normal, but 80% in input is quite high. Would it be worth marking PCR duplicate, or comparing your mapping rates with the public datasets (e.g. ENCODE - I think they had Input as well)?

03-25-2011, 05:50 AM	#13
andreiafonseca Junior Member Location: Portugal Join Date: Mar 2008 Posts: 9	can you explain me what do you mean by marking PCR duplicate?

03-25-2011, 05:59 AM	#14
ttnguyen Member Location: Ireland Join Date: Mar 2010 Posts: 41	This is very good QS I think as the average is high and the variation is low (even though at 3' end). Could you show your Bowtie command?

03-25-2011, 06:03 AM	#15
andreiafonseca Junior Member Location: Portugal Join Date: Mar 2008 Posts: 9	-f -a --best --strata -v 2 hg19 fasta then I selected from these the unique alignments can you tell me what do you mean by marking PCR duplicate?

03-25-2011, 06:14 AM	#17
ttnguyen Member Location: Ireland Join Date: Mar 2010 Posts: 41	I think that is fine when you chose the -v alignment mode since the QS is good.