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
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
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