TY - JOUR
T1 - Software updates in the illumina hiseq platform affect whole-genome bisulfite sequencing
AU - Toh, Hidehiro
AU - Shirane, Kenjiro
AU - Miura, Fumihito
AU - Kubo, Naoki
AU - Ichiyanagi, Kenji
AU - Hayashi, Katsuhiko
AU - Saitou, Mitinori
AU - Suyama, Mikita
AU - Ito, Takashi
AU - Sasaki, Hiroyuki
N1 - Funding Information:
We would like to thank Miho Miyake, Tomomi Akinaga, and Junko Oishi (Kyushu University) for technical assistance and Illumina sequencing. We also thank Wan kin Au Yeung, Daisuke Saito (Kyushu University), Hisato Kobayashi (Tokyo University of Agriculture), and Yasuhiro Yamamoto (National Institute for Basic Biology) for generous support. This work was partially supported by the Core Research for Evolutional Science and Technology (CREST) from the Japan Agency for Medical Research and Development (AMED) and a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) to H.S. (25112010). K.S. is a Japan Society for the Promotion of Science (JSPS) research fellow.
Publisher Copyright:
© 2017 The Author(s).
PY - 2017/1/5
Y1 - 2017/1/5
N2 - Background: Methylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases. Whole-genome bisulfite sequencing (WGBS), such as MethylC-seq and post-bisulfite adaptor tagging sequencing (PBAT-seq), uses the power of high-throughput DNA sequencers and provides genome-wide DNA methylation profiles at single-base resolution. However, the accuracy and consistency of WGBS outputs in relation to the operating conditions of high-throughput sequencers have not been explored. Results: We have used the Illumina HiSeq platform for our PBAT-based WGBS, and found that different versions of HiSeq Control Software (HCS) and Real-Time Analysis (RTA) installed on the system provided different global CpG methylation levels (approximately 5% overall difference) for the same libraries. This problem was reproduced multiple times with different WGBS libraries and likely to be associated with the low sequence diversity of bisulfite-converted DNA. We found that HCS was the major determinant in the observed differences. To determine which version of HCS is most suitable for WGBS, we used substrates with predetermined CpG methylation levels, and found that HCS v2.0.5 is the best among the examined versions. HCS v2.0.12 showed the poorest performance and provided artificially lower CpG methylation levels when 5-methylcytosine is read as guanine (first read of PBAT-seq and second read of MethylC-seq). In addition, paired-end sequencing of low diversity libraries using HCS v2.2.38 or the latest HCS v2.2.58 was greatly affected by cluster densities. Conclusions: Software updates in the Illumina HiSeq platform can affect the outputs from low-diversity sequencing libraries such as WGBS libraries. More recent versions are not necessarily the better, and HCS v2.0.5 is currently the best for WGBS among the examined HCS versions. Thus, together with other experimental conditions, special care has to be taken on this point when CpG methylation levels are to be compared between different samples by WGBS.
AB - Background: Methylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases. Whole-genome bisulfite sequencing (WGBS), such as MethylC-seq and post-bisulfite adaptor tagging sequencing (PBAT-seq), uses the power of high-throughput DNA sequencers and provides genome-wide DNA methylation profiles at single-base resolution. However, the accuracy and consistency of WGBS outputs in relation to the operating conditions of high-throughput sequencers have not been explored. Results: We have used the Illumina HiSeq platform for our PBAT-based WGBS, and found that different versions of HiSeq Control Software (HCS) and Real-Time Analysis (RTA) installed on the system provided different global CpG methylation levels (approximately 5% overall difference) for the same libraries. This problem was reproduced multiple times with different WGBS libraries and likely to be associated with the low sequence diversity of bisulfite-converted DNA. We found that HCS was the major determinant in the observed differences. To determine which version of HCS is most suitable for WGBS, we used substrates with predetermined CpG methylation levels, and found that HCS v2.0.5 is the best among the examined versions. HCS v2.0.12 showed the poorest performance and provided artificially lower CpG methylation levels when 5-methylcytosine is read as guanine (first read of PBAT-seq and second read of MethylC-seq). In addition, paired-end sequencing of low diversity libraries using HCS v2.2.38 or the latest HCS v2.2.58 was greatly affected by cluster densities. Conclusions: Software updates in the Illumina HiSeq platform can affect the outputs from low-diversity sequencing libraries such as WGBS libraries. More recent versions are not necessarily the better, and HCS v2.0.5 is currently the best for WGBS among the examined HCS versions. Thus, together with other experimental conditions, special care has to be taken on this point when CpG methylation levels are to be compared between different samples by WGBS.
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U2 - 10.1186/s12864-016-3392-9
DO - 10.1186/s12864-016-3392-9
M3 - Article
C2 - 28056787
AN - SCOPUS:85008392669
SN - 1471-2164
VL - 18
JO - BMC genomics
JF - BMC genomics
IS - 1
M1 - 31
ER -