2016

2016/7/11-18に、東京大学柏キャンパス(7/11-15)及び東京大学医科学研究所(7/16-18)にて日本学術振興会研究拠点形成事業「MinIONに関する技術講習会」を開催致しました。

Summary

1st Technology Seminar on the MinION sequencing

Title: Serotyping of dengue virus and genotyping of drug resistance genes in malaria parasites using MinION

Background: Recent invent of a portable sequencer, MinION, is expected to change the way of the sequencing analysis drastically. Unlike conventional sequencing platforms, MinION determines the sequence by detecting changes in flow of ion current through an ion channel. Since this is a label-free detection, it requires any optical devices for the signal detection, which would be usually implemented by a large and costive sequencer. Indeed, taking these advantages, MinION has been successfully miniaturized in a portable and one-time use USB device with a reasonable cost of the device itself as well as its consumables. Conversion of a sample to a sequencing template is also simple, consisting solely of adaptor ligation in an isothermal condition, which can be completed within 1-2 hours. One of the biggest advantage in the MinION sequencer lies in the fact that it does not require any installation of conventional laboratory equipment or specific skills in biological experiments in its sequencing procedure. Even electric supply is not always necessary, if the MinION is connected to a laptop PC. These features of MinION have opened the opportunity to enable precise genotyping of pathogens in tropical diseases in rural hospitals or in filed circumstances.

Seminar: We will conduct MinION sequencing using dengue virus and malaria parasites, P. falciparum. The initial goal should be to demonstrate if the serotyping or genotyping of the respective species is possible starting from MinION sequence data. Using the genomic DNA/RNA of the pathogens, it will be amplified using either PCR or other isothermal amplification methods. Using the amplified templates, the sequencing library will be prepared using the sample prep kit provided by Oxford Nanopore Technologies Inc. The sequencing will be run using one to twelve samples per flow cell, expecting approximately 10,000 sequences will be obtained. The obtained sequences will be mapped to the reference genome sequences for each of the serotypes using LAST and BWA, representative sequence alignment programs. The results will be further processed by our in-house prepared bioinformatics software, for which the bench mark test showed the correct separation of the serotypes were possible for >95% of the cases. We will initially start the analysis with in vitro cultured established viral strain and will apply the developed method for the clinical samples. The material viral genomes will be transferred to Japanese team under the condition defined by the appended Material Transfer Agreement. The cost for this study will be covered by a Japanese grant, Asia-Africa Research Exchange Program, JSPS (2016-2019; PI Yutaka Suzuki, University of Tokyo).

Detailed Methods:

Samples and MinION sequencing
The LAMP method will be used to amplify the viruses as previously described with a slight modification. Briefly, 1 µl of patient serum is added to the reaction solution, which contained 9.97 µl of 0.1% Triton-X 100, 0.5 µl of 1 M Tris-HCl (pH8.8), 0.25 µl of 1 M KCl, 1.75 µl of 100 mM MgSO4, 1.4 µl of 25 mM dNTPs, 1 µl of GelGreen 10,000x solution, 0.1 µl of RNase inhibitor, 1 µl of Bst HS polymerase, 0.03 µl of AMV reverse transcriptase, and 2 µl of 2 M trehalose at 63°C. After 90 min of amplification, the amplicons are quantitated with a BioAnalyzer or evaluated by agarose gel electrophoresis.The Genomic DNA Sequencing kit (SQK-MAP-004) is used to prepare the library for MinION sequencing, following the manufacturer’s instructions. LAMP amplicons is end-repaired using the NEBNext End Repair Module (New England Biolabs). The total volume of the 100 µl reaction is incubated at room temperature for 30 minutes and purified using 100 µl of Agencourt Ampure XP beads (Beckman Coulter). The obtained products are used for dA-tailing with the NEBNext dA-tailing module (New England Biolabs) in a total volume of 30 µl at 37°C for 30 minutes. Adaptor ligation is performed using reagents from Oxford Nanopore and the Blunt/TA Ligase Master Mix (New England Biolabs). The reactions are made with the dA-tailed DNA added to 10 µl of adapter mix, 2 µl of HP adapter, 8 µl of molecular grade water and 50 µl of Blunt/TA Ligase Master Mix at room temperature for 10 minutes. The adapter-ligated DNA is purified using 10 µl of Dynabeads His-Tag Isolation & Pulldown (Life Technologies) and wash with buffer from Oxford Nanopore. Samples were eluted in 25 µl of elution buffer from Oxford Nanopore.During the sequencing, MinION Flow Cell (FLO_MAP003) R7.0 or R7.3 are used with the MinION device. Before sequencing, the Flow Cell is primed using 150 mL of EP buffer twice for 10 minutes. The sequencing library mix is prepared by combining 6 µl of library with 140 µl of EP buffer and 4 µl of fuel mix. Then, the library mix is loaded onto the MinION Flow Cell. For the sequencing reaction, the Genomic DNA 48 hour sequencing protocol is used with the MinKNOW software. The base call is made with the Metrichor Agent (https://metrichor.com) using the workflow r7.X 2D Basecalling version 1.24. The base called sequence data in fast5 format are converted to the fastq format and used for subsequent analyses. RT-PCR is performed as described in Supplementary Methods for the validation analysis. Sanger sequencing of the RT-PCR and LAMP products will be conducted using a standard protocol.

Computational procedure
The sequences are mapped to the reference genomes of dengue viruses (NC_001477.1, NC_001474.2, NC_001475.2 and NC_002640.1 for D1, D2, D3 and D4, respectively). To map a sequence, the alignment program LAST (version 5.48) is used with the options ‘-r6 -q12 -a15 -b3 -e150 -m100 -Q1 -j4’ for lastal command and ‘-m1’ for last-split command. Note that with the recently improved version of last-train, we suggest replacing '-a15 -b3' with '-a12 -A15 -b4', which sets a lower cost for deletions than insertions. When necessary, genomic sequences corresponding to the primer sites are masked. The number of mapped reads is counted at the indicated threshold. For instance, when detecting serotypes, any serotypes supported by more than ten sequence tags corresponding to a LAST score >150 are considered. To separate barcoding oligos, the sequences that match multiple barcodes with a LAST score <100 are discarded. Based on the alignments, the most frequent base at each genomic coordinate is selected as a “consensus” for each sample. When the sequence depth do not exceed ten, the base is considered “N”. When calling SNVs are found, the generated consensus sequences are further compared with the reference genomes. A SNV is identified when the sequence reads supporting the variant is twice as large as the rest of the bases taken together. For the SNV calling based on Illumina sequencing, sequencing is conducted using the TruSeq DNA kit on the HiSeq2500 platform at 36 base-single-end. Sequences are aligned using BWA and SNV calls that are made using GATK with the default settings. Statistical significance is evaluated by the indicated methods using statistical analysis software, R (https://www.r-project.org/).

Schedule

Hands on training

July 11th
9:45-12:00 Seminar
13:00-17:00 Hands on training and Sterring meeting
18:00- Welcome party@Doutonbori (Okonomiyaki)
July 12th
9:00-12:00 Hands on training
13:00-17:00 Hands on training
July 13th
9:00-12:00 Informatics seminar
13:00-17:00 Hands on training
July 14th
9:00-12:00 Data analysis
13:00-15:00 Wrap-up meeting
16:30- BBQ Party@Kashiwa Campus
July 15th
9:00-17:30 Symposium
July 16th (Follow up seminar: specific for a mosquito)
13:00-17:00 Hands on training (DNA amplification)
July 17th(Follow up seminar: specific for a mosquito)
9:00-12:00 Hands on training (Template preparation)
13:00-17:00 Hands on training (MinION run)
July 18th (Follow up seminar: specific for a mosquito)
9:00-12:00 Data analysis

Symposium

Date
2016.7.15 Friday

Venue
Environmental Studies Bldg. The University of Tokyo Kashiwa Campas
*Lunch-on-seminar: Bioscienses Bldg. The University of Tokyo Kashiwa Campas

Time table
9:00-9:30 Opening remarks
Yutaka Suzuki(The University of Tokyo, Japan)
Josef Tuda (Sam Ratulangi University, Indonesia)
9:30-10:30 Plenary I (Chair: Yutaka Suzuki)
James Brayer (Oxford Nanopore Technologies, UK)
Wojciech Makałowski(University of Münster, Germany)
Martin Frith (The University of Tokyo, Japan)
10:30-11:00 Break
11:00-12:00 Plenary II (Chair: Junya Yamagishi)
Yutaka Suzuki(The University of Tokyo, Japan)
Yuki Eshita (Hokkaido University, Japan)
Kyoko Hayashida(Hokkaido University, Japan)
12:00-14:00 Lunch-on-seminar (Bioscienses Bldg.)
Sysmex Corporation 14:00-15:00 Workshop I (Chair: Wojciech Makałowski)
Junya Yamagishi(Hokkaido University, Japan)
Ronald Lucky Runtuwene(The University of Tokyo, Japan)
Tadashi Imanishi(Tokai University, Japan)
Takuya Maeda (Saitama Medical University)
15:00-15:30 Spesial Talk
Takashi Mino (The University of Tokyo, Japan)
15:30-16:30 Workshop II (Chair: Yuki Eshita)
Yoshifumi Nishikawa (Obihiro University of Agriculture and Veterinary Medicine, Japan)
Mohammed Tolba(Assuit University, Egypt)
Nguyen Thuy Linh (National Institute of Health and Epidemiology, Vietnam)
Nuankanya Sathirapongsasuti(Mahidol University, Thailand)
Raweewan Srisawat(Mahidol University, Thailand)
16:30-17:00 Open discussion
17:00 Closing remarks
Ryuichiro Maeda(Obihiro University of Agriculture and Veterinary Medicine, Japan)