Biology subjects

Hebert, P. D.

Publications and source records attributed to Hebert, P. D..

5 recordsLinked to original sources

bioRxiv2026

Performance Test of the QNome Nanopore Sequencer

Nanopore sequencers have the potential to liberate DNA sequencing from centralized core facilities to distributed analytical nodes. Until now, Oxford Nanopore Technologies (ONT) has been the sole manufacturer of a portable nanopore sequencer, but analogous platforms are in production. Nanopore sequencers from Qitan Technology (QT) are widely used in China but have been unavailable outside that nation and lack independent performance testing. Enabled by early access to QTs least expensive sequencer and flow cell, the QNome-3841 and QCell-384, we tested whether they could generate accurate DNA barcodes cost-effectively. In several tests involving amplicon pools from 95 to 9,120 specimens, QT recovered valid DNA barcodes from nearly as many specimens (98%) as ONT. QT sequences had slightly lower fidelity than their ONT counterparts and QT frequently failed to resolve the correct length of G/C homopolymers. However, barcode sequences from the two platforms were nearly indistinguishable after bioinformatic treatment. QTs wash kit performed well, enabling a QCell to sequence eight amplicon pools with zero carryover between runs and minimal degradation of the flow cell. Its ultra-fast protocol allowed library preparation in a single step that could be completed in 15 minutes, but this came at the cost of lower quality data. Once widely available, QT devices will be well-suited for supporting DNA barcode analysis.

genomics↗

bioRxiv2025

Multiple variants of the mitochondrial COI DNA barcode region are prevalent in North European sawflies

DNA barcoding, the use of standard segments of DNA to assign specimens to a species, has emerged as a major field of biodiversity research over the last 20 years. Large-scale global initiatives are building DNA barcode reference libraries for animals, fungi, and plants, while pipelines are being developed for metabarcoding-based biomonitoring. The effectiveness of these approaches rests on the premise that much less variation exists within species than between them. While exceptions occur, this principle has been demonstrated to apply in the many animal taxa where the barcode region of the COI gene is effective in species discrimination. Sawflies are an exception to this general pattern because DNA barcodes often fail to distinguish congeneric species, an observation which prompted us to search for an explanation. Using high-throughput single-molecule DNA sequencing to recover COI sequences from thousands of sawflies, we found that single individuals often possess multiple, seemingly functional, full-length DNA barcodes - a phenomenon not documented at similar prevalence in any animal taxon. While the evolutionary causes of multiple variants require further investigation, our observation is remarkable as it violates the one-barcode-one-specimen assumption. The presence of multiple variants of barcodes within individuals does not jeopardize the concept, but its occurrence does introduce a complexity for species inventories based on metabarcoding. They will overestimate the species count when barcode-based operational species units are used as species proxies. Similarly, reference libraries must consider how best to deal with the high frequency of multiple variants in sawflies and any other groups of organisms. Significance StatementDNA barcoding is revolutionizing biodiversity science by enabling the accurate identification of organisms, accelerating taxonomic workflows, and permitting DNA-based biomonitoring. The DNA barcode region for the animal kingdom, mitochondrial COI, is highly effective in discriminating species in almost all studied animal groups. However, the use DNA barcoding is sometimes complicated by the presence of nuclear pseudogenes (NUMTs) or by variants of the mitogenome itself (heteroplasmy) within individuals. By using high-throughput sequencing (HTS) to analyze thou-sands of specimens, we demonstrate that multiple, seemingly functional, full-length variants of the COI barcode region are frequent in North European sawflies. Since these variants are sometimes very divergent, it is important to consider the impact of this within-individual variability on studies based on DNA barcodes.

evolutionary biology↗

bioRxiv2020

Massive Multiplexing Can Deliver a $1 Test for COVID-19

The severe acute respiratory syndrome virus, SARS-CoV-2 (hereafter COVID-19), rapidly achieved global pandemic status, provoking large-scale screening programs in many nations. Their activation makes it imperative to identify methods that can deliver a diagnostic result at low cost. This paper describes an approach which employs sequence variation in the gene coding for its envelope protein as the basis for a scalable, inexpensive test for COVID-19. It achieves this by coupling a simple RNA extraction protocol with low-volume RT-PCR, followed by E-Gel screening and sequencing on high-throughput platforms to analyze 10,000 samples in a run. Slight modifications to the protocol could support screening programs for other known viruses and for viral discovery. Just as the $1,000 genome is transforming medicine, a $1 diagnostic test for viral and bacterial pathogens would represent a major advance for public health.

genomics↗

bioRxiv2018

Revealing the Complexities of Metabarcoding with a Diverse Arthropod Mock Community

DNA metabarcoding is an attractive approach for monitoring biodiversity. However, it is subject to biases that often impede detection of all species in a sample. In particular, the proportion of sequences recovered from each species depends on its biomass, mitome copy number, and primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 BINs, a species proxy. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body partitions (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicon generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, other variables did. As expected, the best recovery was obtained from the Single Leg treatment, but the Bulk Abdomen produced a more uniform read abundance than the Bulk Leg or Composite Leg samples. Primer choice also influenced species recovery. Our results reveal how variation in protocols can have substantive impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote. Although metabarcoding is a powerful approach, further optimization of analytical protocols is crucial to obtain reproducible results and increase its cost-effectiveness.

ecology↗

bioRxiv2017

A Sequel to Sanger: Amplicon Sequencing That Scales

Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system. By examining templates from more than 5,000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL reduces costs 40-fold from Sanger analysis. Reflecting the capacity of each instrument to recover sequences from more than five million DNA extracts a year, this platform facilitates massive amplicon characterization.

molecular biology↗