MinION Nanopore Library Preparation

Amplified cDNA from Round B was purified using AMPure XP beads (Beckman Coulter, Brea, CA), and 1 μg DNA was used as input into Oxford Nanopore Genomic DNA MAP-003 Kits (Chik1, Ebola1) or MAP-004 Kits (HepC1, Ebola2) for generation of MinION Oxford Nanopore-compatible libraries [9 (link), 11 (link)]. Briefly, the steps include: (1) addition of control lambda phage DNA, (2) end-repair with the NEBNext End Repair Module, (3) 1× AMPure purification, (4) dA-tailing with the NEBNext dA-tailing Module, (5) ligation to protein-linked adapters HP/AMP (Oxford Nanopore Technologies, Oxford, UK) using the NEBNext QuickLigation Module for 10 min at room temperature, (6) purification of ligated libraries using magnetic His-Tag Dynabeads (Life Technologies), and (7) elution in 25 μL buffer (Oxford Nanopore Technologies). Lambda phage DNA was not added during preparation of the Ebola2 sample library.

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Greninger A.L., Naccache S.N., Federman S., Yu G., Mbala P., Bres V., Stryke D., Bouquet J., Somasekar S., Linnen J.M., Dodd R., Mulembakani P., Schneider B.S., Muyembe-Tamfum J.J., Stramer S.L, & Chiu C.Y. (2015). Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis. Genome Medicine, 7, 99.

Publication 2015

AMPure XP beads His-Tag Dynabeads

Buffer Cdna Lambda phage Library Ligation Linked protein

Corresponding Organization :

Other organizations : University of California, San Francisco, National Institute of Biomedical Research, Hologic (United States), Global Viral, Abbott (United States), American Red Cross, Metabiota (United States)

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Variable analysis

independent variables

Amplified cDNA from Round B was purified using AMPure XP beads
1 μg DNA was used as input into Oxford Nanopore Genomic DNA MAP-003 Kits (Chik1, Ebola1) or MAP-004 Kits (HepC1, Ebola2)

dependent variables

Generation of MinION Oxford Nanopore-compatible libraries

control variables

Addition of control lambda phage DNA
End-repair with the NEBNext End Repair Module
1× AMPure purification
DA-tailing with the NEBNext dA-tailing Module
Ligation to protein-linked adapters HP/AMP using the NEBNext QuickLigation Module for 10 min at room temperature
Purification of ligated libraries using magnetic His-Tag Dynabeads
Elution in 25 μL buffer

positive controls

Addition of control lambda phage DNA

negative controls

Lambda phage DNA was not added during preparation of the Ebola2 sample library

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