TY - JOUR
T1 - Evaluation of clinical formalin-fixed paraffin-embedded tissue quality for targeted-bisulfite sequencing
AU - Ohmomo, Hideki
AU - Komaki, Shohei
AU - Ono, Kanako
AU - Sutoh, Yoichi
AU - Hachiya, Tsuyoshi
AU - Arai, Eri
AU - Fujimoto, Hiroyuki
AU - Yoshida, Teruhiko
AU - Kanai, Yae
AU - Sasaki, Makoto
AU - Shimizu, Atsushi
N1 - Funding Information:
This study was supported by the Japan Agency for Medical Research and Development (AMED) under Grant Number JP17km0105004.
Publisher Copyright:
© 2020 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd
PY - 2021/2
Y1 - 2021/2
N2 - Formalin-fixed paraffin-embedded (FFPE) tissues are promising biological resources for genetic research. Recent improvements in DNA extraction from FFPE samples allowed the use of these tissues for multiple sequencing methods. However, fundamental research addressing the application of FFPE-derived DNA for targeted-bisulfite sequencing (TB-seq) is lacking. Here, we evaluated the suitability of FFPE-derived DNA for TB-seq. We conducted TB-seq using FFPE-derived DNA and corresponding fresh frozen (FF) tissues of patients with kidney cancer and compared the quality of DNA, libraries, and TB-seq statistics between the two preservation methods. The approximately 600-bp average fragment size of the FFPE-derived DNA was significantly shorter than that of the FF-derived DNA. The sequencing libraries constructed using FFPE-derived DNA and the mapping ratio were approximately 10 times and 10% lower, respectively, than those constructed using FF-derived DNA. In the mapped data of FFPE-derived DNA, duplicated reads accounted for > 60% of the obtained sequence reads, with lower mean on-target coverage. Therefore, the standard TB-seq protocol is inadequate for obtaining high-quality data for epigenetic analysis from FFPE-derived DNA, and technical improvements are necessary for enabling the use of archived FFPE resources.
AB - Formalin-fixed paraffin-embedded (FFPE) tissues are promising biological resources for genetic research. Recent improvements in DNA extraction from FFPE samples allowed the use of these tissues for multiple sequencing methods. However, fundamental research addressing the application of FFPE-derived DNA for targeted-bisulfite sequencing (TB-seq) is lacking. Here, we evaluated the suitability of FFPE-derived DNA for TB-seq. We conducted TB-seq using FFPE-derived DNA and corresponding fresh frozen (FF) tissues of patients with kidney cancer and compared the quality of DNA, libraries, and TB-seq statistics between the two preservation methods. The approximately 600-bp average fragment size of the FFPE-derived DNA was significantly shorter than that of the FF-derived DNA. The sequencing libraries constructed using FFPE-derived DNA and the mapping ratio were approximately 10 times and 10% lower, respectively, than those constructed using FF-derived DNA. In the mapped data of FFPE-derived DNA, duplicated reads accounted for > 60% of the obtained sequence reads, with lower mean on-target coverage. Therefore, the standard TB-seq protocol is inadequate for obtaining high-quality data for epigenetic analysis from FFPE-derived DNA, and technical improvements are necessary for enabling the use of archived FFPE resources.
KW - DNA methylation
KW - Epigenetics
KW - Formalin-fixed paraffin-embedded tissue
KW - Fresh frozen tissue
KW - Targeted-bisulfite sequencing
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U2 - 10.1111/pin.13054
DO - 10.1111/pin.13054
M3 - Article
C2 - 33333623
AN - SCOPUS:85097609262
VL - 71
SP - 135
EP - 140
JO - Acta Pathologica Japonica
JF - Acta Pathologica Japonica
SN - 1320-5463
IS - 2
ER -