A comparison of Oxford nanopore library strategies for bacterial genomics

Type Article
Date 2023-10
Language English
Author(s) Sauvage Thomas1, Cormier AlexandreORCID2, Passerini DelphineORCID1
Affiliation(s) 1 : Ifremer, MASAE Microbiologie Aliment Santé Environnement, F-44000, Nantes, France
2 : Ifremer, IRSI-SeBiMER, F-29280, Plouzané, France
Source Bmc Genomics (1471-2164) (Springer Science and Business Media LLC), 2023-10 , Vol. 24 , N. 1 , P. 627 (16p.)
DOI 10.1186/s12864-023-09729-z
WOS© Times Cited 1
Keyword(s) Hybrid, Native, Leakage, Ligation, Mapping, minION, Tagmentation, Tandem, Transposase, Replicons
Abstract

Background Oxford nanopore Technologies (ONT) provides three main library preparation strategies to sequence bacterial genomes. These include tagmentation (TAG), ligation (LIG) and amplification (PCR). Despite ONT’s recommendations, making an informed decision for preparation choice remains difficult without a side-by-side comparison. Here, we sequenced 12 bacterial strains to examine the overall output of these strategies, including sequencing noise, barcoding efficiency and assembly quality based on mapping to curated genomes established herein. Results Average read length ranged closely for TAG and LIG (> 5,000 bp), while being drastically smaller for PCR (< 1,100 bp). LIG produced the largest output with 33.62 Gbp vs. 11.72 Gbp for TAG and 4.79 Gbp for PCR. PCR produced the most sequencing noise with only 22.7% of reads mappable to the curated genomes, vs. 92.9% for LIG and 87.3% for TAG. Output per channel was most homogenous in LIG and most variable in PCR, while intermediate in TAG. Artifactual tandem content was most abundant in PCR (22.5%) and least in LIG and TAG (0.9% and 2.2%). Basecalling and demultiplexing of barcoded libraries resulted in ~ 20% data loss as unclassified reads and 1.5% read leakage. Conclusion The output of LIG was best (low noise, high read numbers of long lengths), intermediate in TAG (some noise, moderate read numbers of long lengths) and less desirable in PCR (high noise, high read numbers of short lengths). Overall, users should not accept assembly results at face value without careful replicon verification, including the detection of plasmids assembled from leaked reads.

Full Text
File Pages Size Access
Publisher's official version 16 2 MB Open access
Additional file 1. Preparation kits. 1 423 KB Open access
Additional file 2. Fail vs. Pass reads. 2 651 KB Open access
Additional file 3. Assembly details and chromosome attributes. 4 584 KB Open access
Additional file 4. Barcoding statistics. 5 605 KB Open access
Additional file 5. DNA extraction protocol. 1 415 KB Open access
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