Application of the MinION sequencer for identifying several bacteria pathogens

Università degli Studi di Sassari Dipartimento di Scienze Biomediche Master Universitario Internazionale di II livello denominato International Master in Medical Biotechnology in Accordo la Huè University of Medicine and Pharmacy, (Huè, Vietnam) (Direttore del Master Prof Daniele Dessì) TITLE “APPLICATION OF THE MINION SEQUENCER FOR IDENTIFYING SEVERAL BACTERIA PATHOGENS” Relatore: Assoc Prof RODNEY A LEA Dr NGUYEN HOANG BACH Assoc Prof NGUYEN DANG QUOC CHAN Anno Accademico 2019/2020 Tesi di: HO THI THANH MAI MINISTRY OF EDUCATION AND TRAINING HUE UNIVERSITY HUE UNIVERSITY OF MEDICINE AND PHARMACY HO THI THANH MAI “APPLICATION OF THE MINION SEQUENCER FOR IDENTIFYING SEVERAL BACTERIA PATHOGENS” THESIS OF MASTER IN MEDICAL SCIENCE HUẾ - 2020 ACKNOWLEDGEMENTS Firstly, I would like to thank Associate Professor Rodney A Lea, Doctor Nguyen Hoang Bach, Associate Professor Nguyen Dang Quoc Chan, my supervisors who guided, supported and helped me for my Master study and research with all kindness, patience and immense knowledge Associate Professor Rodney is also a person that gives me a chance to go abroad to Australia I really appreciated him My sincere thanks also goes to Professor Flavia She is an expert about bacterial genetics at QIMR, Australia She had given me appropriate knowledge in order to make me understand more about my projects I would like to express my special thanks of gratitude to Doctor Neven who is very kind and enthusiastic She spent her time to guide me about anything sequencing A grateful thanks to send Associate Professor Larisa for providing me with all the facility that was required I also thank Ms Cao Thi Thao Van, Mr Nguyen Hoang Son and all colleges in Innovation Health Biomedical I, Queensland University of Technology, Australia I am extremely thankful to thank Associate Professor Nguyen Dang Quoc Chan, director of School Medicine and Pharmacy, the University of Da Nang, who gave me a change to attend this Master course Special thanks to all of the Microbiology Department : Associate Professor Le Van An, Associate Professor Le Viet Quynh Tram, Ms Nguyen Thi Tuyet Ngoc who helped me and shared useful experiences for my research Lastly, I would like to thank my family: my parents, my husband and my daughter, for supporting me through the time that I learnt my Master course Ho Thi Thanh Mai i ABBREVIATIONS DNA Deoxyribonucleic acid dsDNA Double-stranded DNA ssDNA Single-stranded DNA dNTP Deoxynucleotide triphosphate ddNTP Dideoxynucleotide triphosphate gDNA Genomic DNA HGP Human Genome Project NGS Next Generation Sequencing ONT Oxford Nanopore Technologies PCR Polymerase chain reaction RT PCR Real time Polymerase chain reaction SGS Second generation sequencing SS Sanger Sequencing TGS Third generation sequencing RNA Ribonucleic acid rRNA Riboxom ribonucleic acid WGS Whole-genome sequencing ARMA Antibiotic Resistance Mapping Application WIMP What is my pot MS Mass spectrometry SMRT Single molecule real time PacBio Pacific Biosciences MALDI-TOF Matrix-assisted laser desorption ionization time-of-flight ii ABSTRACT Infectious diseases causes by bacterial pathogens that have a substantial global health impact and it could become serious if remained undiagnosed with several diseases related to nervous system, blood infection,…Many technologies in diagnostics are related to bacteria culture performed by morphological trait differentiation and biochemical testing, which could be more time-consuming and labor intensive In this study, we offer using a culture-independent analysis simultaneously for several bacteria pathogens from clinical samples using MinION sequencing Oxford Nanopore Technologies (ONT) recently released the MinION in 2014, giving it the smallest footprint of any current sequencing platform and can be connected to an individual computer via a USB 3.0-interface The MinION is unique among TGS platforms in that it is the only portable real time device for DNA and RNA sequencing Importantly, the MinION is commercially available at a very low entry cost of $1,000USD For this research, whole genomes of bacterial pathogens will be extracted from urine and sputum samples of 10 patients in Hue University Hospital, Viet Nam The bacterial DNA concentration were estimated by using a quantitative PCR assay (qPCR) 16S rRNA gene of bacteria were sequenced on Minion device (Oxford Nanopore) by using 16S barcoding kit SQKRAB204 on R9.4 flow cell Genomic 16S rRNA will be analysed using external databases and bioinformatics pipelines with MinKNOW, EPI2ME software On the EPI2ME platform, the bacteria pathogens base on the total of reads were classified via “analysis workflow” as: FASTQ 16S QC Barcoding, FASTQ WIMP (What in my pot ) MinION sequencing was optimized to 98.7 % workflow successful with 2,160,000 reads analysed ; 1,884,027 reads classified, the average of iii sequencing length was 1,340 base for pathogen detection compared with other methods ( culture-dependent method, real-time PCR, AFB Ziehl-Neelsen stain) The species levels of bacteria could identify accurately via WIMP program on EPI2ME Nanopore 16S results showed good correlation with bacteria culture, real-time PCR but can be more sensitive, manageable, less expensive and time saving Sequencing the genomic 16S rRNA via MinION sequencer could be a good ideal in clinical diagnostic microbiology With the rapid development of Nanopore technology, in the term of chemistry advances, more accurate base callers and analysis software, the MinION offers promise for wide use in the future and might contribute to a reduction in broad spectrum antibiotic use iv TÓM TẮT Các bệnh truyền nhiễm vi khuẩn gây có ảnh hưởng sức khỏe tồn cầu đáng kể trở nên nghiêm trọng khơng chẩn đốn nhanh chóng với số bệnh liên quan đến hệ thống thần kinh, nhiễm trùng máu….Tuy nhiên, công nghệ chẩn đốn liên quan đến ni cấy vi khuẩn thực phương pháp phân biệt hình thái xét nghiệm sinh hóa, tốn nhiều thời gian công sức Trong nghiên cứu này, đề nghị sử dụng phân tích độc lập ni cấy số tác nhân vi khuẩn từ mẫu lâm sàng sử dụng cơng nghệ giải trình tự gen MinION Gần đây, công nghệ Oxford Nanopore (ONT) cho đời thiết bị Minion vào năm 2014, mang lại cho dấu ấn nhỏ tảng giải trình tự kết nối với máy tính thơng qua giao diện USB 3.0 MinION số tảng NGS chỗ thiết bị thời gian thực di động để giải trình tự DNA RNA Điều quan trọng, MinION có sẵn thị trường với chi phí thấp khoảng $ 1.000USD Đối với nghiên cứu này, toàn bộ gen vi khuẩn tách chiết từ mẫu nước tiểu mẫu đàm 10 bệnh nhân Bệnh viện Đại học Y Dược Huế, Việt Nam Nồng độ DNA vi khuẩn định lượng nhanh cách sử dụng kỹ thuật qPCR Vùng gen16S rRNA vi sinh vật giải trình tự thiết bị giải trình tự MinION (Oxford Nanopore) cách sử dụng mã vạch 16S kit có tên SQK-RAB204 flow cell R9.4 Vùng gen 16S rRNA phân tích sở liệu xử lý tin sinh học với phần mềm MinKNOW, EPI2ME Trên phần mềm EPI2ME, số loài vi khuẩn định danh dựa vào “reads” phân loại thơng qua quy trình phân tích như: FASTQ 16S QC Barcode, FASTQ WIMP v Phương pháp giải trình tự MinION tối ưu hóa đạt 98.7% mức độ làm việc với 2,160,000 “reads” phân tích; 1,884,027 “reads” phân lớp chiều dài trung bình gen 1,340 base để phát vi khuẩn so sánh với phương pháp khác ( nuôi cấy, RT PCR, Nhuộm AFB Ziehl-Neelsen) Vi khuẩn định danh xác mức độ lồi thơng qua chương trình WIMP phần mềm EPI2ME Các kết chạy qua lỗ Nanopore gene 16S mối tương quan tốt với phương pháp nuôi cấy vi khuẩn thơng thường nhạy hơn, dễ quản lý, tốn tiết kiệm nhiều thời gian Vùng gen 16S rRNA giải trình tự gen thiết bị MinION ý tưởng tốt chẩn đoán vi sinh lâm sàng Với phát triển nhanh chóng cơng nghệ Nanopore , cải tiến hóa học, phần mềm phân tích gọi base xác hơn, MinION hứa hẹn sử dụng rộng rãi tương lai góp phần giảm thiểu việc sử dụng kháng sinh phổ rộng vi CONTENTS ACKNOWLEDGEMENTS i ABSTRACT iii TÓM TẮT v LIST OF FIGURES .ix LIST OF TABLES xi INTRODUCTION CHAPTER I REVIEW OF LITERATURE 1.1 Introduction about microbiology laboratory diagnostic 1.1.1 Phenotypic method 1.1.1.1 Cytological examination 1.1.1.2 Culture methods 1.1.2 Genotype method 12 1.1.2.1 Real-Time Polymerase chain reaction (RT- PCR) 12 1.1.2.2 Sequence technology 14 1.2 Introduction about 16S rRNA gene sequencing for identification bacteria pathogens 24 1.2.1 Characteristics of 16S rRNA 24 1.2.2 Comparison of the sequencing platforms for 16S metagenomic analysis to identify bacteria 25 1.3 Introduction about 16S Barcoding kit (SQK-RAB204) 28 CHAPTER II MATERIALS AND METHODS 29 2.1 Ethics statement 29 2.2 Sampling 29 2.3 DNA extraction 29 2.4 Optimization of qPCR for detection of 16S ribosomal rRNA of bacteria 30 vii 2.5 MinION sequencing 30 2.5.1 Library preparation 31 2.5.2 Sequencing 32 2.5.3 Analysis data 35 CHAPTER III RESULTS 37 3.1 Species identification by using culture-dependent method and Real time PCR 37 3.2 Optimization of qPCR assays for detection of gen 16S rRNA bacteria 38 3.3 Evaluation of Nanopore MinION sequencing result 39 3.3.1 Control sequencing with the MinION 39 3.3.2 16S QC Barcoding 41 3.3.3 WIMP for identifying bacteria pathogens 45 CHAPTER IV DISCUSSION 50 CONCLUSION 53 REFERENCES 54 viii LIST OF FIGURES Figure Classification of general Positive Gram and Negative Gram Bacteria Figure Gram stain Figure Ziehl–Neelsen stain Figure Mycobacterium tuberculosis visualization using the Ziehl–Neelsen stain Figure Biomedical test Figure MALDI-TOF MS for bacterial identification 11 Figure Review of molecular biology methods used for the identification of different microorganisms 13 Figure The Sanger (chain-termination) method for DNA sequencing 16 Figure NGS Workflow 18 Figure 10 The Minion sequencing device 20 Figure 11 The Oxford Nanopore sequencing process 22 Figure 12 A representation of 16S rRNA 24 Figure 13 Most common metabarcoding sequencing strategies for each sequencing technology generation 26 Figure 14 Workflow for the SQK RAB-204 kit 28 Figure 15 Detecting structural variants with nanopore 34 Figure 16 The default data analysis workflow 35 Figure 17 Data acquisition 36 Figure 18 Mux Scan Results 39 Figure 19 A screenshot of the flow cell activity during day run .40 Figure 20 Cumulative Output Result 40 Figure 21 The workflow successful of 16S QC Barcoding 41 Figure 22 Read count per hour result 43 Figure 23 The chart of yield per flow cell 43 ix Figure 24 The distribution of quality score 44 Figure 25 The sequence length (bases) 44 Figure 26 The reads count per Barcode ID 45 Figure 27 WIMP process results 46 Figure 28 The Taxa at species level 46 Figure 29 NCBI Taxonomy tree (WIMP) 47 x LIST OF TABLES Table Summary of Acid-Fast Stain Table Comparison of the available sequencing platforms for 16S metagenomic analysis using metabarcoding approach 27 Table Bacterial identification by Ziehl-Neelsen stain and RT - PCR 37 Table Bacterial identification by culture-dependent method from urine samples 37 Table Result of qPCR running 38 Table The number of reads for each Barcode 42 Table Comparing Minion sequencing results and the other methods 48 Table Comparing the culture-dependent method and MinION sequencing results 49 xi ...MINISTRY OF EDUCATION AND TRAINING HUE UNIVERSITY HUE UNIVERSITY OF MEDICINE AND PHARMACY HO THI THANH MAI ? ?APPLICATION OF THE MINION SEQUENCER FOR IDENTIFYING SEVERAL BACTERIA PATHOGENS? ?? THESIS OF. .. simultaneously for several bacteria pathogens from clinical samples using MinION sequencing Oxford Nanopore Technologies (ONT) recently released the MinION in 2014, giving it the smallest footprint of any... microbiology With the rapid development of Nanopore technology, in the term of chemistry advances, more accurate base callers and analysis software, the MinION offers promise for wide use in the future