why are all DiffBind tutorial's ATAC-seq peak intervals 400 bp for all intervals?
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3 months ago
mrj ▴ 90

I have followed DiffBind tutorial https://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf I have found out that the returned count matrix's peak intervals are always 400bp. Literally, all the peak intervals in the count matrix are 400 bp. I am wondering why it is happening.

this was the case for my own data as well.

My code is as follows.

library(DiffBind)
samples <- read.csv("tamoxifen.csv")
DBdata1 <- dba(sampleSheet=samples)
DBA <- dba.count(DBdata1,score=DBA_SCORE_READS)
counts <- dba.peakset(DBA, bRetrieve=T, DataType=DBA_DATA_FRAME)

when I inspect the count matrix returned in the above code, it looks like the following

CHR  START END       BT4741 ...
chr18  90841  91241    2
chr18  111395 111795  21

If you subtract END from START, you will get 400bp. The answer is the same for the entire dataset.

peak ATAC-seq intervals DiffBind consensus • 764 views
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3 months ago
ATpoint 57k

See section 3.3. in the manual:

(...) As this example is based on a transcription factor that binds to the DNA, resulting in "punctate", relatively narrow peaks, the default option to re-center each peak around the point of greatest enrichment is appropriate. This keeps the peaks at a consistent width (in this case, the default summits=200 results in 401bp-wide intervals, extending 200bp up- and downstream of the summit)

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Thank you for this answer.

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Could you share the number of width you set in your ATAC-seq analysis? I have read the manual of Diffbind, however, I still do not know the suitable value for the width. Many thanks to you~

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5 weeks ago
Rory Stark ★ 1.3k

Buries in the docs (?DiffBind3) is a recommendation to use summits=100 for ATAC-seq, which results in 201bp windows.

It helps to look sat the distribution of fragment lengths in your data. If you've done paired-end sequencing and trimmed adaptors, you'll get a variety of sequence lengths. Generally in a good quality ATAC experiment most of the fragments will be around 50bp-100bp. If that is the case, the summits parameter can be set even lower, e.g. summits=50 or summits=75.

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