Single end read WGBS

Notorious ATG · 09-12-2019, 02:55 PM

Hi all,

We study DNA methylation in mouse tissues, usually with extremely small input quantities of DNA (i.e. total yields of 5-15ng) from FACS-sorted microdissected brain tissues. Our sequencing center uses the tagmentation-based library preparation protocol described in Wang et al., (2013).

The downside of this is that despite our efforts to adjust tagmentation time/concentration, we always end up with really small insert sizes. When we do paired-end sequencing, almost all of the second read goes to waste because it overlaps the first.

A cheaper option that seemingly eliminates this redundancy is to do 200bp single-end reads. We have a quote for that which would seemingly meet our coverage requirements and is much cheaper than our usual paired-end method.

My question: is this a good way to go? Are there any obvious pitfalls I should be aware of when using 200bp single-end reads for WGBS (one advantage is that these are inbred, genetically identical mice, so no indels to worry about).

Thank you!

Wang, Q., Gu, L., Adey, A., Radlwimmer, B., Wang, W., Hovestadt, V., … Weichenhan, D. (2013). Tagmentation-based whole-genome bisulfite sequencing. Nature Protocols, 8(10), 2022–2032. https://doi.org/10.1038/nprot.2013.118

fkrueger · 09-13-2019, 06:52 AM

In my opinion long single-end reads are a very good alternative to overlapping, redundant PE reads. Read processing should also be generally faster.

Just make sure that the reads are properly quality and adapter trimmed (which will almost certainly be an issue with reads this long), a default run through e.g. Trim Galore should do the trick. Good luck!

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09-12-2019, 02:55 PM	#1
Notorious ATG Junior Member Location: Houston Join Date: Mar 2016 Posts: 3	Single end read WGBS Hi all, We study DNA methylation in mouse tissues, usually with extremely small input quantities of DNA (i.e. total yields of 5-15ng) from FACS-sorted microdissected brain tissues. Our sequencing center uses the tagmentation-based library preparation protocol described in Wang et al., (2013). The downside of this is that despite our efforts to adjust tagmentation time/concentration, we always end up with really small insert sizes. When we do paired-end sequencing, almost all of the second read goes to waste because it overlaps the first. A cheaper option that seemingly eliminates this redundancy is to do 200bp single-end reads. We have a quote for that which would seemingly meet our coverage requirements and is much cheaper than our usual paired-end method. My question: is this a good way to go? Are there any obvious pitfalls I should be aware of when using 200bp single-end reads for WGBS (one advantage is that these are inbred, genetically identical mice, so no indels to worry about). Thank you! Wang, Q., Gu, L., Adey, A., Radlwimmer, B., Wang, W., Hovestadt, V., … Weichenhan, D. (2013). Tagmentation-based whole-genome bisulfite sequencing. Nature Protocols, 8(10), 2022–2032. https://doi.org/10.1038/nprot.2013.118

09-13-2019, 06:52 AM	#2
fkrueger Senior Member Location: Cambridge, UK Join Date: Sep 2009 Posts: 625	In my opinion long single-end reads are a very good alternative to overlapping, redundant PE reads. Read processing should also be generally faster. Just make sure that the reads are properly quality and adapter trimmed (which will almost certainly be an issue with reads this long), a default run through e.g. Trim Galore should do the trick. Good luck!