Enrichment post-library preparation enhances the sensitivity of high-throughput sequencing-based detection and characterization of viruses from complex samples

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

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Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing

Biochimie ◽

10.1016/j.biochi.2012.10.025 ◽

2013 ◽

Vol 95 (4) ◽

pp. 743-750 ◽

Cited By ~ 30

Author(s):

Yuanyuan Ren ◽

Lei Chen ◽

Yiyun Zhang ◽

Xiangyang Kang ◽

Zhiyi Zhang ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Populus Tomentosa ◽

Identification And Characterization

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Characterization of the Fecal Microbiota Using High-Throughput Sequencing Reveals a Stable Microbial Community during Storage

PLoS ONE ◽

10.1371/journal.pone.0046953 ◽

2012 ◽

Vol 7 (10) ◽

pp. e46953 ◽

Cited By ~ 119

Author(s):

Ian M. Carroll ◽

Tamar Ringel-Kulka ◽

Jennica P. Siddle ◽

Todd R. Klaenhammer ◽

Yehuda Ringel

Keyword(s):

Microbial Community ◽

High Throughput ◽

High Throughput Sequencing ◽

Fecal Microbiota

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Extraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling

PLoS ONE ◽

10.1371/journal.pone.0254971 ◽

2021 ◽

Vol 16 (11) ◽

pp. e0254971

Author(s):

Federico Rossi ◽

Alessandro Crnjar ◽

Federico Comitani ◽

Rodrigo Feliciano ◽

Leonie Jahn ◽

...

Keyword(s):

Dna Methylation ◽

Tree Ring ◽

High Throughput ◽

Environmental Conditions ◽

High Throughput Sequencing ◽

Wood Formation ◽

Whole Genome ◽

Library Preparation ◽

Bisulfite Treatment ◽

Standing Trees

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.

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Molecular characterization of a new soybean-infecting member of the genus Nepovirus identified by high-throughput sequencing

Archives of Virology ◽

10.1007/s00705-016-3152-9 ◽

2016 ◽

Vol 162 (4) ◽

pp. 1089-1092 ◽

Cited By ~ 4

Author(s):

Tuba Yasmin ◽

Berlin D. Nelson ◽

Houston A. Hobbs ◽

Nancy K. McCoppin ◽

Kris N. Lambert ◽

...

Keyword(s):

Molecular Characterization ◽

High Throughput ◽

High Throughput Sequencing

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High-throughput sequencing identification and characterization of potentially adhesion-related small RNAs in Streptococcus mutans

Journal of Medical Microbiology ◽

10.1099/jmm.0.000718 ◽

2018 ◽

Vol 67 (5) ◽

pp. 641-651 ◽

Cited By ~ 5

Author(s):

Wenhui Zhu ◽

Shanshan Liu ◽

Jia Liu ◽

Yan Zhou ◽

Huancai Lin

Keyword(s):

Streptococcus Mutans ◽

High Throughput ◽

Small Rnas ◽

High Throughput Sequencing ◽

Identification And Characterization

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Characterization of microbial community in cold-chain hairtail fish by high-throughput sequencing technology

Journal of Food Protection ◽

10.4315/jfp-20-393 ◽

2021 ◽

Author(s):

Jiali Xing ◽

Xiaorong Xu ◽

Xiaohu Luo ◽

Ruihang Zheng ◽

Lingyan Mao ◽

...

Keyword(s):

Microbial Community ◽

High Throughput ◽

High Throughput Sequencing ◽

Bacterial Flora ◽

Diversity Indices ◽

Cold Chain ◽

Class Level ◽

The Family ◽

Dominant Bacteria

Abstract: High-throughput sequencing was used to analyze the microbial communities in the muscle samples of hairtail fish to study their diversity and dynamic changes during cold-chain circulation. The results showed that the richness and diversity of the microbial community in hairtail fish had a transient decline in 0–24 h and decreased after the first rise during 24–216 h. The diversity and richness of bacteria in cold-chain hairtail fish reached the maximum at 168 h. The Shannon and Simpson diversity indices of the bacteria were 2.96 and 0.16, respectively, and their ACE and Chao1 richness indices were 254.84 and 155.10, respectively. In addition, the dominant bacteria were Proteobacteria in the phylum level, Gammaproteobacteria in the class level, Pseudomonadales in the order level, Pseudomonadaceae in the family level, and Pseudomonas in the genus level, and their relative abundance were 80.52%, 72.11%, 76.68%, 23.25%, and 53.50%, respectively. In this study, the structure of bacterial flora and the dominant bacteria in cold-chain hairtail fish were analyzed by high-throughput sequencing to provide a basis for exploring how to maintain the freshness of hairtail fish and for predicting the shelf-life of hairtail fish.

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