MinION sequencing - SRA

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ERX926229: MinION sequencing
1 OXFORD_NANOPORE (MinION) run: 4,555 spots, 11.9M bases, 12.3Mb downloads

Submitted by: CE-M-M-, RESEARCH CENTER FOR MOLECULAR MEDICINE OF THE AUSTRIAN ACADEMY OF SCIENCES (CE-M-M-, RESEARCH CENTER FOR MOLECULAR MEDICINE OF)

Study: Sequencing of LCMV Clone 13 genomic fragment from the late stage of infection

PRJEB9029 • ERP010083 • All experiments • All runs

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RNA viruses represent a unique model for evolutionary biology, because of extremly high error rate (10^-3 substitution per site), small genomes and large population sizes. Lymphocytic choriomeningitis virus (LCMV) is a prototypic member for Arenaviruses, that serves as a model system for multiple virological studies. This virus infects preferentially Rodents. LCMV genome consists of two single stranded RNA molecules (L - large and S - small segments). MinION provided by Oxford Nanopore Technologies, allows to get a single-molecule resolution, that makes it a valuable tool to reconstruct viral evolution both, in vivo and in vitro. We sequenced a 1.8 Kb fragment of the L segment using MinION (Oxford Nanopore Technologies, Oxford, UK) obtained from the tissue of infected C57BL/6J mouse at the late stage of infection and from the plasmid encoding viral L segment LCMV. This approach could potentially provide us with a more complete understanding of viral evolution process.

Sample: Plasmid

SAMEA3334664 • ERS700203 • All experiments • All runs

Organism: Mammarenavirus choriomeningitidis

Library:

Name: Plasmid

Instrument: MinION

Strategy: AMPLICON

Source: GENOMIC

Selection: PCR

Layout: SINGLE

Construction protocol: The Genomic DNA Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK) provided as part of the MinION early access program (MAP) along with user-supplied reagents were used to generate a sequence-ready library for the MinION sequencing procedure. Gel-purified PCR products obtained either from cDNA of spleen tissue of C57BL/6J mice at 50 day post infection with LCMV Clone 13 or from a plasmid encoding the L segment of LCMV Clone 13 were used for library preparation (2 individual samples in total) according Amplicon Sequencing using Genomic DNA Sequencing Kit (SQK-MAP003) protocol (Version DP003_3001_revD_05Sep2014, last modified on Nov 06, 2014).

Experiment attributes: (show all 6 attributes...) (hide...)

kit: SQK-MAP003

sequencing protocol: gDNA

dna extraction protocol: RNA extraction with TRIzol�, Life Technologies acc (show full text...)(hide...)

RNA extraction with TRIzol�, Life Technologies according the standard protocol followed by cDNA synthesis with sequence-specific primer (SuperScript II Reverse Transcriptase, Life Technologies) and PCR amplification with Phusion Hot Start II High-Fidelity DNA Polymerase, Life Technologies. The obtained PCR product was visualized staining with ethidium bromide by gel electrophoresis in agarose gel and isolated with QIAquick Gel Extraction Kit, QIAGEN according the manufacturer protocol. DNA concentration was measured with Qubit dsDNA HS (High Sensitivity) Assay Kit, Life Technologies

reloading of reagents: Each sample was sequenced for 2 hours, the flowcel (show full text...)(hide...)

Each sample was sequenced for 2 hours, the flowcell was washed between 2 runs with Wash Kit provided by Oxford Nanopore Technologies, Oxford, UK according the manufacturer protocol and 30 minutes run was performed before the loading of second sample.

library_source_details: Plasmid encoding the L segment of Clone 13 LCMV

library_selection_details: PCR amplification from plasmid encoding L segment (show full text...)(hide...)

PCR amplification from plasmid encoding L segment LCMV Clone 13 using high fidelity polymerase

Runs: 1 run, 4,555 spots, 11.9M bases, 12.3Mb

Run	# of Spots	# of Bases	Size	Published
ERR845318	4,555	11.9M	12.3Mb	2016-04-02

ID:: 2402919

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