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Evaluation of hybrid sequencing by comparing DNA digestion fragment pattern of Xbal, S1 nuclease in pulsed field gel electrophoresis (PFGE) and in in silico analysis of whole genome sequencing (WGS) data

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Abstract
최근 많은 유전체 분석 연구에 차세대 시퀀싱(next generation sequencing, NGS)이 적용되고 있으며 그 중요성에 대해서 보고되고 있다. 미생물의 유전자에 관련된 생명과학 연구에 전장 유전체 시퀀싱(whole genome sequencing, WGS)의 적용이 활발해지고 있으며, 그에 따라 sequencing 기술의 개발과 정확도에도 관심이 커지고 있다. 또한, sequencing 정확도를 높이기 위해서 hybrid sequencing을 하거나 추가 실험을 진행하여 비교, 분석하는 시도도 이루어지고 있다. 본 실험에서는 hybrid sequencing으로 WGS 데이터를 생산했으며, PFGE를 진행하여 그 결과를 비교함으로써 유사도를 확인했다. 펄스 필드 겔 전기영동(Pulse field gel electrophoresis, PFGE)은 XbaI 제한효소와 S1 nuclease를 적용하여 molecular typing과 plasmid profiling을 진행했다. XbaI 제한효소를 적용한 WGS 데이터와 PFGE 데이터의 molecular typing결과에서 샘플의 수가 적거나 여러 그룹으로 재분류되는 두 가지 그룹을 제외하고 모든 그룹에서 cluster가 유사하게 나타났다. 하지만, plasmid profiling의 경우에는 유사하게 나타나지 않았다. 이는 패턴으로 cluster를 비교할 때에는 만들어질 수 있는 패턴의 다양성, 그룹에 속한 샘플의 수, 그룹 내의 샘플정보의 일치가 결과 값에 영향을 끼친다는 것을 나타낸다. 또한 ANI와 PFGE 패턴의 비교 결과가 유사하게 나타난 샘플 간에 유사한 크기의 contig와 band가 72%로 확인되었다. 즉, 이는 WGS data와 PFGE 데이터 간에 28% 정도의 차이가 나타남을 암시한다. 본 연구를 통한 결과들을 바탕으로 WGS 데이터가 PFGE 패턴과 완벽히 일치하지 않다는 것을 확인하였으며 보다 정확한 비교와 분석을 위해서는 추가적인 연구가 필요하다고 사료된다.|Recently, next generation sequencing (NGS) has been applied to many genome analysis studies, and its importance has been reported. The application of whole genome sequencing (WGS) to life science research related to microbial genes is becoming more active, and interest in the development and accuracy of sequencing technology is also growing. Accordingly, to improve sequencing accuracy, attempts are being made to conduct and analyze hybrid sequencing or compare with additional experiments. In this experiment, WGS data was produced by hybrid sequencing, and similarity was confirmed by additionally performing PFGE. Pulse field gel electrophoresis (PFGE) was performed for molecular typing and plasmid profiling by applying XbaI restriction enzyme and S1 nuclease. In the molecular typing results of WGS data and PFGE data to which XbaI restriction enzyme was applied, clusters were similar in all groups except for two groups where the number of samples was small or reclassified into several groups. However, result of plasmid profiling did not appear similarly. This indicates that when comparing clustering by pattern, the diversity of patterns that can be made, the number of samples belonging to a group, and the matching of sample information in a group affect the result value. In addition, contigs and bands of similar size were identified at 78% among samples with similar ANI and PFGE pattern comparison results. That is, this implies that a difference of about 22% appears between the WGS data and the PFGE data. Based on the results of this study, it was confirmed that the WGS data did not completely match the PFGE results, and additional research is needed for more accurate comparison and analysis.
Author(s)
Lee Sun woo
Issued Date
2023
Awarded Date
2023-02
Type
Dissertation
URI
https://dcoll.jejunu.ac.kr/common/orgView/000000011073
Alternative Author(s)
이선우
Affiliation
Graduate School Jeju National University
Department
대학원 분자생명공학전공
Advisor
Unno Tatsuya
Table Of Contents
CONTENT 1
LIST OF FIGURES 2
LIST OF TABLES 3
ABSTRACT 4
INTRODUCTION 6
MATERIAL AND METHOD 8
Sample information 8
Molecular typing and plasmid profiling using PFGE 8
DNA extraction and sequencing 10
WGS data analysis 11
Patterning of WGS data and Gel photos 11
Statistical analysis 11
RESULT AND DISCUSSION 12
Comparison of XbaI/PFGE results and WGS data 12
Comparison of PFGE and ANI results 17
Comparison of PFGE band and WGS data contig 23
SUPPLEMENTARY INFORMATION 25
CONCLUSION 35
REFERENCE 36
ACKNOWLEDGEMENT 39
Degree
Master
Publisher
Graduate School Jeju National University
Citation
Lee Sun woo. (2023). Evaluation of hybrid sequencing by comparing DNA digestion fragment pattern of Xbal, S1 nuclease in pulsed field gel electrophoresis (PFGE) and in in silico analysis of whole genome sequencing (WGS) data.
Appears in Collections:
Faculty of Biotechnology > Molecular Biotechnology
공개 및 라이선스
  • 공개 구분공개
  • 엠바고2023-02-17
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