High‐Throughput Sequencing of PCR Products Tagged with Universal Primers Using 454 Life Sciences Systems

In a search for viral infections, double-stranded RNA (dsRNA) were recovered from a diseased cyclamen (Cyclamen persicum Mill.) accession (Cic) and analyzed by high-throughput sequencing (HTS) technology. Analysis of the HTS data showed the presence of Fig mosaic emaravirus (FMV) in this accession. The complete sequences of six FMV-Cic RNA genomic segments were determined from the HTS data and using Sanger sequencing. All FMV-Cic RNA segments are similar in size to those of FMV from fig (FMV-Gr10), with the exception of RNA-6 that is one nucleotide longer. The occurrence of FMV in cyclamen was investigated through a small-scale survey, from which four plants (out of 18 tested) were found RT-PCR positive. To study sequence variations of cyclamen isolates of FMV, RT-PCR products generated through the amplification of the partially RNA-dependent RNA polymerase (RdRp, RNA-1), glycoprotein (GP, RNA-2), and nucleocapsid (NCP, RNA-3) genes were explored. The nucleotide sequence identities for cyclamen isolates ranged between 86% and 99% in RNA-1, 93% and 99% in RNA-2, and 98% and 99% in RNA-3, while lower identity levels were observed with the sequences of fig isolates. Based on the phylogenetic tree obtained with a 304-nt fragment of RNA3, all FMV isolates from cyclamens were assigned to a single cluster close to fig isolates from the Mediterranean. FMV was graft-transmitted to healthy cyclamens eliciting symptoms similar to those observed in the Cic accession, thus suggesting a causal role of FMV in the symptoms that prompted the investigation. This is the first report of FMV in a non-fig host, Cyclamen persicum, a finding that may help in the control of the mosaic and mosaic-like diseases of fig and cyclamen, respectively.

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High-throughput sequencing of kDNA amplicons for the analysis ofLeishmaniaminicircles and identification of Neotropical species

Parasitology ◽

10.1017/s0031182017002013 ◽

2017 ◽

Vol 145 (5) ◽

pp. 585-594 ◽

Cited By ~ 7

Author(s):

ARTHUR KOCHER ◽

SOPHIE VALIÈRE ◽

ANNE-LAURE BAÑULS ◽

JÉRÔME MURIENNE

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Pcr Primers ◽

Genomic Region ◽

Kinetoplast Dna ◽

Sequence Comparisons ◽

Conserved Sequence ◽

Species Complexes ◽

Pcr Products ◽

Minicircle Sequence

SUMMARYLeishmaniakinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitiveLeishmaniadetection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization ofLeishmaniaminicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18Leishmaniastrains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis ofLeishmaniarelationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications ofLeishmaniaspp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications inLeishmaniaresearch.

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CoronaHiT: high-throughput sequencing of SARS-CoV-2 genomes

Genome Medicine ◽

10.1186/s13073-021-00839-5 ◽

2021 ◽

Vol 13 (1) ◽

Cited By ~ 3

Author(s):

Dave J. Baker ◽

Alp Aydin ◽

Thanh Le-Viet ◽

Gemma L. Kay ◽

Steven Rudder ◽

...

Keyword(s):

Maximum Likelihood ◽

Genome Sequencing ◽

High Throughput ◽

High Throughput Sequencing ◽

Rapid Expansion ◽

Library Preparation ◽

Method Performance ◽

Genome Coverage ◽

Maximum Likelihood Tree ◽

Pcr Products

AbstractWe present CoronaHiT, a platform and throughput flexible method for sequencing SARS-CoV-2 genomes (≤ 96 on MinION or > 96 on Illumina NextSeq) depending on changing requirements experienced during the pandemic. CoronaHiT uses transposase-based library preparation of ARTIC PCR products. Method performance was demonstrated by sequencing 2 plates containing 95 and 59 SARS-CoV-2 genomes on nanopore and Illumina platforms and comparing to the ARTIC LoCost nanopore method. Of the 154 samples sequenced using all 3 methods, ≥ 90% genome coverage was obtained for 64.3% using ARTIC LoCost, 71.4% using CoronaHiT-ONT and 76.6% using CoronaHiT-Illumina, with almost identical clustering on a maximum likelihood tree. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally.

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Barcoding the food chain: from Sanger to high-throughput sequencing

Genome ◽

10.1139/gen-2016-0028 ◽

2016 ◽

Vol 59 (11) ◽

pp. 946-958 ◽

Cited By ~ 11

Author(s):

Joanne E. Littlefair ◽

Elizabeth L. Clare

Keyword(s):

Dna Barcoding ◽

High Throughput ◽

Food Chain ◽

High Throughput Sequencing ◽

Human Mobility ◽

Environmental Dna ◽

Universal Primers ◽

Individual Species ◽

Parasitic Infections ◽

Forensic Evaluation

Society faces the complex challenge of supporting biodiversity and ecosystem functioning, while ensuring food security by providing safe traceable food through an ever-more-complex global food chain. The increase in human mobility brings the added threat of pests, parasites, and invaders that further complicate our agro-industrial efforts. DNA barcoding technologies allow researchers to identify both individual species, and, when combined with universal primers and high-throughput sequencing techniques, the diversity within mixed samples (metabarcoding). These tools are already being employed to detect market substitutions, trace pests through the forensic evaluation of trace “environmental DNA”, and to track parasitic infections in livestock. The potential of DNA barcoding to contribute to increased security of the food chain is clear, but challenges remain in regulation and the need for validation of experimental analysis. Here, we present an overview of the current uses and challenges of applied DNA barcoding in agriculture, from agro-ecosystems within farmland to the kitchen table.

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