Express barcoding with NextGenPCR and MinION for species-level sorting of ecological samples

Vasiliţa, Cristina; Feng, Vivian; Hansen, Anker; Hartop, Emily; Srivathsan, Amrita; Struijk, Robert B.; Meier, Rudolf

doi:10.1101/2023.04.27.538648

Cited by 3 publications

(12 citation statements)

References 27 publications

(29 reference statements)

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“…To further speed up the process of estimating the species diversity in a bulk sample, real‐time barcoding can be coupled with the “express barcoding” of Vasilita et al. (2023) that accelerates amplicon production by using NextGenPCR® thermocyclers. These cyclers use special polymerases and fast heating/cooling in three preset heating zones.…”

Section: Discussionmentioning

confidence: 99%

“…3 h of sequencing on a Flongle. A re‐analysis of the dataset using a super‐accuracy model for reads accumulated during the first 3 h shows that the overall success rate only drops by 2% compared with analysing all reads for a 24 h run (Vasilita et al., 2023: Table 1). Thus, users can make informed choices on which coverage is needed for any particular project.…”

Section: Discussionmentioning

confidence: 99%

“…For phorids, a 313 bp COI fragment was amplified as described in Vasilita et al. (2023). The standard 13 bp tags that we have been routinely using for MinION barcodes as designed for Srivathsan et al.…”

Section: Methodsmentioning

confidence: 99%

“…Two sets of amplicons were used to assess the performance of the latest chemistry and real-time barcoding using MinION. The first set consisted of COI amplicons for 7679 spider specimens while the second consisted of amplicons for 285 phorid specimens also evaluated in Vasilita et al (2023). The spider DNA was extracted using 20 lL of HotSHOT (total volume = 40 lL after addition of 20 lL neutralization buffer; Truett et al, 2000).…”

Section: Pcr With Tagged Primersmentioning

confidence: 99%

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ONTbarcoder 2.0: rapid species discovery and identification with real‐time barcoding facilitated by Oxford Nanopore R10.4

Srivathsan,

Feng,

Suárez

et al. 2023

Cladistics

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Most arthropod species are undescribed and hidden in specimen‐rich samples that are difficult to sort to species using morphological characters. For such samples, sorting to putative species with DNA barcodes is an attractive alternative, but needs cost‐effective techniques that are suitable for use in many laboratories around the world. Barcoding using the portable and inexpensive MinION sequencer produced by Oxford Nanopore Technologies (ONT) could be useful for presorting specimen‐rich samples with DNA barcodes because it requires little space and is inexpensive. However, similarly important is user‐friendly and reliable software for analysis of the ONT data. It is here provided in the form of ONTbarcoder 2.0 that is suitable for all commonly used operating systems and includes a Graphical User Interface (GUI). Compared with an earlier version, ONTbarcoder 2.0 has three key improvements related to the higher read quality obtained with ONT's latest flow cells (R10.4), chemistry (V14 kits) and basecalling model (super‐accuracy model). First, the improved read quality of ONT's latest flow cells (R10.4) allows for the use of primers with shorter indices than those previously needed (9 bp vs. 12–13 bp). This decreases the primer cost and can potentially improve PCR success rates. Second, ONTbarcoder now delivers real‐time barcoding to complement ONT's real‐time sequencing. This means that the first barcodes are obtained within minutes of starting a sequencing run; i.e. flow cell use can be optimized by terminating sequencing runs when most barcodes have already been obtained. The only input needed by ONTbarcoder 2.0 is a demultiplexing sheet and sequencing data (raw or basecalled) generated by either a Mk1B or a Mk1C. Thirdly, we demonstrate that the availability of R10.4 chemistry for the low‐cost Flongle flow cell is an attractive option for users who require only 200–250 barcodes at a time.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Pcr With Tagged Primersmentioning

confidence: 99%

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ONTbarcoder 2.0: rapid species discovery and identification with real‐time barcoding facilitated by Oxford Nanopore R10.4

Srivathsan,

Feng,

Suárez

et al. 2023

Cladistics

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“…The data are available from https:// figsh are. com/s/ 5a4c6 a9615 fe71a d8284 (Vasilita et al, 2023).…”

Section: Data Ava I L a B I L I T Y S Tat E M E N Tmentioning

confidence: 99%

Express barcoding with NextGenPCR and MinION for species‐level sorting of ecological samples

Vasilita,

Feng,

Hansen

et al. 2024

Molecular Ecology Resources

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The use of DNA barcoding is well established for specimen identification and large‐scale biodiversity discovery, but remains underutilized for time‐sensitive applications such as rapid species discovery in field stations, identifying pests, citizen science projects, and authenticating food. The main reason is that existing express barcoding workflows are either too expensive or can only be used in very well‐equipped laboratories by highly‐trained staff. We here show an alternative workflow combining rapid DNA extraction with HotSHOT, amplicon production with NextGenPCR thermocyclers, and sequencing with low‐cost MinION sequencers. We demonstrate the power of the approach by generating 250 barcodes for 285 specimens within 6 h including specimen identification through BLAST. The workflow required only the following major equipment that easily fits onto a lab bench: Thermocycler, NextGenPCR, microplate sealer, Qubit, and MinION. Based on our results, we argue that simplified barcoding workflows for species‐level sorting are now faster, more accurate, and sufficiently cost‐effective to replace traditional morpho‐species sorting in many projects.

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Rapid species discovery and identification with real-time barcoding facilitated by ONTbarcoder 2.0 and Oxford Nanopore R10.4

Srivathsan

Feng

Suárez

et al. 2023

Preprint

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Most animal species are undescribed and hidden in biodiversity samples consisting of thousands of specimens that are difficult to sort to species using morphological characters. Sorting with DNA barcodes is an attractive alternative, but needs cost-effective techniques that are suitable for use in many laboratories around the world so that barcoding can be made widely available. Oxford Nanopore's portable and inexpensive MinION sequencers are a good option because they allow for reducing the sequencing and capital-cost of a fully functional barcoding laboratory. We here provide an update to the MinION barcoding software ONTbarcoder that is available for commonly used operating systems and includes a GUI. We show that the read quality obtained with ONT's latest basecallers and chemistry (R10.4 flowcells, Q20+ kits) allow for three key improvements. Firstly, ONTbarcoder can now deliver real-time barcoding to complement ONT's real-time sequencing. This means that the first barcodes are obtained within minutes of starting a sequencing run and a significant proportion of barcodes are available before sequencing completes. This real-time feature of ONTbarcoder can be configured to handle both Mk1B and Mk1C. The input is a demultiplexing sheet and base-called or raw data. Secondly, the improved read quality of ONT's latest flow cells (R10.4) allows for the use of primers with shorter indices than what was previously needed (9bp vs. 12-13bp). This decreases primer cost and improves PCR success rates. Thirdly, we demonstrate that the availability of R10.4 chemistry in the low-cost Flongle flowcell is an attractive option for users who require only 200-250 barcodes at a time.

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Express barcoding with NextGenPCR and MinION for species-level sorting of ecological samples

Cited by 3 publications

References 27 publications

ONTbarcoder 2.0: rapid species discovery and identification with real‐time barcoding facilitated by Oxford Nanopore R10.4

ONTbarcoder 2.0: rapid species discovery and identification with real‐time barcoding facilitated by Oxford Nanopore R10.4

Express barcoding with NextGenPCR and MinION for species‐level sorting of ecological samples

Rapid species discovery and identification with real-time barcoding facilitated by ONTbarcoder 2.0 and Oxford Nanopore R10.4

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