Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments.
Bisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplificatio...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2016-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4816320?pdf=render |
| _version_ | 1818368496991469568 |
|---|---|
| author | Gordon R McInroy Dario Beraldi Eun-Ang Raiber Katarzyna Modrzynska Pieter van Delft Oliver Billker Shankar Balasubramanian |
| author_facet | Gordon R McInroy Dario Beraldi Eun-Ang Raiber Katarzyna Modrzynska Pieter van Delft Oliver Billker Shankar Balasubramanian |
| author_sort | Gordon R McInroy |
| collection | DOAJ |
| description | Bisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification. The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis. Here, we report a method that enables more accurate methylation analysis, by rebuilding bisulfite-damaged components of a DNA library. This recovery after bisulfite treatment (ReBuilT) approach enables PCR-free bisulfite sequencing from low nanogram quantities of genomic DNA. We apply the ReBuilT method for the first whole methylome analysis of the highly AT-rich genome of Plasmodium berghei. Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias. Our method will be widely applicable for quantitative methylation analysis, even for technically challenging genomes, and where limited sample DNA is available. |
| first_indexed | 2024-12-13T23:08:53Z |
| format | Article |
| id | doaj.art-d883e1b98cc8430b9e1160d9e3f25378 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-13T23:08:53Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-d883e1b98cc8430b9e1160d9e3f253782022-12-21T23:28:10ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01113e015232210.1371/journal.pone.0152322Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments.Gordon R McInroyDario BeraldiEun-Ang RaiberKatarzyna ModrzynskaPieter van DelftOliver BillkerShankar BalasubramanianBisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification. The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis. Here, we report a method that enables more accurate methylation analysis, by rebuilding bisulfite-damaged components of a DNA library. This recovery after bisulfite treatment (ReBuilT) approach enables PCR-free bisulfite sequencing from low nanogram quantities of genomic DNA. We apply the ReBuilT method for the first whole methylome analysis of the highly AT-rich genome of Plasmodium berghei. Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias. Our method will be widely applicable for quantitative methylation analysis, even for technically challenging genomes, and where limited sample DNA is available.http://europepmc.org/articles/PMC4816320?pdf=render |
| spellingShingle | Gordon R McInroy Dario Beraldi Eun-Ang Raiber Katarzyna Modrzynska Pieter van Delft Oliver Billker Shankar Balasubramanian Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments. PLoS ONE |
| title | Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments. |
| title_full | Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments. |
| title_fullStr | Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments. |
| title_full_unstemmed | Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments. |
| title_short | Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments. |
| title_sort | enhanced methylation analysis by recovery of unsequenceable fragments |
| url | http://europepmc.org/articles/PMC4816320?pdf=render |
| work_keys_str_mv | AT gordonrmcinroy enhancedmethylationanalysisbyrecoveryofunsequenceablefragments AT darioberaldi enhancedmethylationanalysisbyrecoveryofunsequenceablefragments AT eunangraiber enhancedmethylationanalysisbyrecoveryofunsequenceablefragments AT katarzynamodrzynska enhancedmethylationanalysisbyrecoveryofunsequenceablefragments AT pietervandelft enhancedmethylationanalysisbyrecoveryofunsequenceablefragments AT oliverbillker enhancedmethylationanalysisbyrecoveryofunsequenceablefragments AT shankarbalasubramanian enhancedmethylationanalysisbyrecoveryofunsequenceablefragments |