Comparison of Next-Generation Sequencing Systems

Liu, Lin; Li, Yinhu; Li, Siliang; Hu, Ni; He, Yingkun; Pong, Ray; Lin, Danni; Lü, Lihua; Law, Maggie

doi:10.1155/2012/251364

Cited by 1,048 publications

(612 citation statements)

References 27 publications

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“…In the present study, culture-based microbiological criteria were coupled with culture-independent methods for in-depth understanding of the overall structure of the vaginal bacterial biota. The approach based on Ion Torrent PGM sequencing of the 16S rRNA gene is a novel high-throughput method with a faster turnaround than other next-generation sequencing techniques [43,44]. Application of the Ion Torrent technology was suitable for the investigation of the bacterial ecology in the BV vaginal ecosystem, allowing us to detect its compositional change at phylum, genus, and species level.…”

Section: Discussionmentioning

confidence: 99%

Bacterial biota of women with bacterial vaginosis treated with lactoferrin: an open prospective randomized trial

Pino

Giunta

Randazzo

et al. 2017

Microbial Ecology in Health and Disease

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Background: Bacterial vaginosis is the most frequent condition associated to the vaginal microbiota imbalance, affecting about the 40–50% of women in the world. Even if antibiotics are effcetive for bacterial vaginosis treatment a long-term recurrence rates, higher than 70%, is recorded. Lactoferrin is an iron-binding glycoprotein with bacteriostatic and bactericidal properties. It owns the ability to protect the host against infection, by binding and regulating the iron needed for the bacterial proliferation.Objective: The present study was an open prospective randomized trial (registration no. SHI-EVE-2014.01) aimed at characterizing the bacterial biota of women affected by bacterial vaginosis (BV) and assessing the effects of two different lactoferrin concentrations (100 mg and 200 mg vaginal pessaries) on the composition and dynamics of the vaginal bacterial biota.Design: Sixty women with BV were recruited and randomized into two groups to receive lactoferrin pessaries for 10 days. Clinical evaluation was based on Amsel criteria and Nugent scores. Culture-dependent methods and Ion Torrent PGM sequencing of the 16S rRNA gene were applied to study in depth the overall structure of the vaginal bacterial biota and its dynamics during the treatment.Results: Vaginal lactoferrin administration modified the vaginal microbiota composition in patients with BV. During treatment, both 100 mg and 200 mg lactoferrin vaginal pessaries significantly decreased the occurrence of bacteria associated with BV, such as Gardnerella, Prevotella, and Lachnospira, and increased the occurrence of Lactobacillus species. The bacterial biota balance was maintained up to 2 weeks after treatment only in women treated with 200 mg lactoferrin pessaries.Conclusions: This study indicates that lactoferrin could be proposed as an alternative therapeutic approach for BV. Our data showed, for the first time, the dominance of Lactobacillus helveticus species during and after vaginal lactoferrin treatment.

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Section: Discussionmentioning

confidence: 99%

Bacterial biota of women with bacterial vaginosis treated with lactoferrin: an open prospective randomized trial

Pino

Giunta

Randazzo

et al. 2017

Microbial Ecology in Health and Disease

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“…The adapters attached to the DNA fragment to be sequenced is further attached to beads and clonally amplified by emPCR [17]. These clonally amplified DNA attached to beads are deposited on glass plate where the beads get covalently bound [28,30].…”

Section: Sequencing By Hybridization and Ligation (Sbl)mentioning

confidence: 99%

“…To sequence the skipped positions, another round of sequencing occurs with a primer that has one or more bases shorter than the previous primer. Thus, the sequence of complete target DNA can be obtained using anchors of various length [28,30].…”

Section: Sequencing By Hybridization and Ligation (Sbl)mentioning

confidence: 99%

“…Short read lengths lead to inaccuracy in read assembly as it is difficult to assemble with any pipeline and additionally more time is required for sequencing [30]. However modified platform for sequencing by hybridization has been developed that will be dealt in third generation sequencing technologies (section on Complete Genomic technology).…”

Section: Sequencing By Hybridization and Ligation (Sbl)mentioning

confidence: 99%

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High Throughput Sequencing: An Overview of Sequencing Chemistry

et al. 2016

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“…By contrast, the PacBio System (Pacific Biosciences, Menlo Park, California) offers long read lengths in the order of 12 kb with the single molecule real‐time (SMRT) sequencing technology 11. This method provides a fast sample preparation and turnover rate as well as phased sequences 12. One of the limitations of SMRT sequencing is the relatively high error rate (>10%) at the individual read level 13, 14.…”

Section: Introductionmentioning

confidence: 99%

Dual redundant sequencing strategy: Full‐length gene characterisation of 1056 novel and confirmatory HLA alleles

Albrecht

Zweiniger

Surendranath

et al. 2017

HLA

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The high‐throughput department of DKMS Life Science Lab encounters novel human leukocyte antigen (HLA) alleles on a daily basis. To characterise these alleles, we have developed a system to sequence the whole gene from 5′‐ to 3′‐UTR for the HLA loci A, B, C, DQB1 and DPB1 for submission to the European Molecular Biology Laboratory – European Nucleotide Archive (EMBL‐ENA) and the IPD‐IMGT/HLA Database. Our workflow is based on a dual redundant sequencing strategy. Using shotgun sequencing on an Illumina MiSeq instrument and single molecule real‐time (SMRT) sequencing on a PacBio RS II instrument, we are able to achieve highly accurate HLA full‐length consensus sequences. Remaining conflicts are resolved using the R package DR2S (Dual Redundant Reference Sequencing). Given the relatively high throughput of this strategy, we have developed the semi‐automated web service TypeLoader, to aid in the submission of sequences to the EMBL‐ENA and the IPD‐IMGT/HLA Database. In the IPD‐IMGT/HLA Database release 3.24.0 (April 2016; prior to the submission of the sequences described here), only 5.2% of all known HLA alleles have been fully characterised together with intronic and UTR sequences. So far, we have applied our strategy to characterise and submit 1056 HLA alleles, thereby more than doubling the number of fully characterised alleles. Given the increasing application of next generation sequencing (NGS) for full gene characterisation in clinical practice, extending the HLA database concomitantly is highly desirable. Therefore, we propose this dual redundant sequencing strategy as a workflow for submission of novel full‐length alleles and characterisation of sequences that are as yet incomplete. This would help to mitigate the predominance of partially known alleles in the database.

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Comparison of Next-Generation Sequencing Systems

Cited by 1,048 publications

References 27 publications

Bacterial biota of women with bacterial vaginosis treated with lactoferrin: an open prospective randomized trial

Bacterial biota of women with bacterial vaginosis treated with lactoferrin: an open prospective randomized trial

High Throughput Sequencing: An Overview of Sequencing Chemistry

Dual redundant sequencing strategy: Full‐length gene characterisation of 1056 novel and confirmatory HLA alleles

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