MinION sequencing; Acinetobacter sp. ADP1 whole genome sequencing usi... - SRA

ERX719729: MinION sequencing; Acinetobacter sp. ADP1 whole genome sequencing using MinIon R7 chemistry
1 OXFORD_NANOPORE (MinION) run: 9,241 spots, 21.4M bases, 15.3Mb downloads

Design: Acinetobacter baylyi ADP1 genomic DNA was prepared from overnight liquid cultures grown in MAS (Medium for Acinetobacter Supplemented) broth at 30°C with shaking to an O.D.600 of approximately 1.5. Cells were pelleted and lysed in the presence of lysozyme. Genomic DNA was purified by phenol-chloroform phase extraction. Extracted DNA was resolved in 100 μL TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]) supplemented with 10 μg/mL RNase (Sigma, St. Louis, MO, USA). Genomic DNA (5 µg) was diluted in 150 µL Elution Buffer (EB, Tris HCl 10 mM; Qiagen, Hilden, Germany). The DNA sample was then loaded into a G-Tube (Covaris) and centrifuged at 3,300 x g for 1 min. The G-Tube was then inverted and centrifuged again for 1 min. Fragmented DNA profiles were evaluated using a DNA12000 LabChip kit on an Agilent Bioanalyzer and quantified using a Qubit Fluorometer (Life Technologies, Carlsbad, CA, USA). One microgram DNA was end repaired in 100-µL reactions using the NEBNext End-Repair module (New England Biolabs) according to the manufacturer’s instructions. The resulting blunt-ended DNA was cleaned-up using 1 µl AMPure XP bead purification according to the manufacturer’s instructions (Beckmann Coulter Genomics) and eluted in 28 µL EB. A-tailing was performed on 25 µL of the DNA using the NEBNext dA-tailing module (New England Biolabs) in a total volume of 30 µL according to the manufacturer’s instructions. The Genomic DNA Sequencing Kit, SQK-MAP-002 (Oxford Nanopore Technologies Ltd, Oxford, UK), was used to generate MinION® sequencing libraries. Fifty microliters of Blunt/TA ligase master mix (NEB), 10 µL adapter mix, and 10 µL HP adaptor were added to each dA-tailed DNA and incubated at 20°C for 10 min. The two libraries were cleaned up using 0.4 µl volumes of AMPure XP beads according to the manufacturer’s instructions, with the exception that only a single wash was carried out using the wash buffer supplied with the kit. The samples were then eluted in 25-µL elution buffer supplied with the Genomic DNA Sequencing Kit. Ten microliters of tether (Genomic DNA Sequencing Kit) was added and incubated for 10 min at 20°C. Last, 15 µL of HP motor (Genomic DNA Sequencing Kit) was added and incubated overnight at 20°C, giving a total library volume of 50 µL. A new MinIONTM Flow Cell was removed from storage at 4°C, fitted to the MinION® device and held in place with the supplied plastic screws to ensure a good thermal contact. One hundred and fifty microliters of EP Buffer (Genomic DNA Sequencing Kit) was loaded into the sample loading port and left for 10 min to prime the flowcell. The priming process was repeated a second time. Then, 12 µL of library and 4 µL of Fuel Mix (Genomic DNA Sequencing Kit) were added to 134 µL of EP Buffer (Genomic DNA Sequencing Kit) and loaded into the sample loading port of the MinIONTM Flow Cell. The loading was repeated 6, 24, and 30 h after the beginning of the run. Read event data generated by MinKNOW™ control software (version 0.45.3.9) were base-called using the software Metrichor™ (version 0.17).

Submitted by: Genoscope (GSC)

Study: Genome assembly using Nanopore-guided Long and Error-free DNA Reads

PRJEB8723 • ERP009748 • All experiments • All runs

show Abstracthide Abstract

In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments. The assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting a variable error rate. Here, we present a hybrid approach developed to take advantage of data generated using Oxford Nanopore and Illumina sequencing technologies. We sequenced the Acinetobacter baylyi ADP1 genome and applied our method to obtain a nearly perfect (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads. Our method provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, it is already useful in the generation of high-quality genome assemblies.

Sample: DNA sample prepared for sequencing on nanopore technology

SAMEA3283859 • ERS671488 • All experiments • All runs

Organism: Acinetobacter baylyi ADP1

Library:

Name: AWK_ONT_8Kb_library_R7

Instrument: MinION

Strategy: WGS

Source: GENOMIC

Selection: RANDOM

Layout: SINGLE

Construction protocol: The Genomic DNA Sequencing Kit, SQK-MAP-002 (Oxford Nanopore Technologies Ltd, Oxford, UK), was used to generate MinION® sequencing libraries. Fifty microliters of Blunt/TA ligase master mix (NEB), 10 µL adapter mix, and 10 µL HP adaptor were added to each dA-tailed DNA and incubated at 20°C for 10 min. The two libraries were cleaned up using 0.4 µl volumes of AMPure XP beads according to the manufacturer’s instructions, with the exception that only a single wash was carried out using the wash buffer supplied with the kit. The samples were then eluted in 25-µL elution buffer supplied with the Genomic DNA Sequencing Kit. Ten microliters of tether (Genomic DNA Sequencing Kit) was added and incubated for 10 min at 20°C. Last, 15 µL of HP motor (Genomic DNA Sequencing Kit) was added and incubated overnight at 20°C, giving a total library volume of 50 µL.

Spot descriptor:

1 forward8001 reverse

Runs: 1 run, 9,241 spots, 21.4M bases, 15.3Mb

Run	# of Spots	# of Bases	Size	Published
ERR776851	9,241	21.4M	15.3Mb	2015-04-23

ID:: 1459357

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