Highly comparable metabarcoding results from MGI-Tech and Illumina sequencing platforms

With the developments in DNA nanoball sequencing technologies and the emergence of new platforms, there is an increasing interest in their performance in comparison with the widely used sequencing-by-synthesis methods. Here, we test the consistency of metabarcoding results from DNBSEQ-G400RS (DNA nanoball sequencing platform by MGI-Tech) and NovaSeq 6000 (sequencing-by-synthesis platform by Illumina) platforms using technical replicates of DNA libraries that consist of COI gene amplicons from 120 soil DNA samples. By subjecting raw sequencing data from both platforms to a uniform bioinformatics processing, we found that the proportion of high-quality reads passing through the filtering steps was similar in both datasets. Per-sample operational taxonomic unit (OTU) and amplicon sequence variant (ASV) richness patterns were highly correlated, but sequencing data from DNBSEQ-G400RS harbored a higher number of OTUs. This may be related to the lower dominance of most common OTUs in DNBSEQ data set (thus revealing higher richness by detecting rare taxa) and/or to a lower effective read quality leading to generation of spurious OTUs. However, there was no statistical difference in the ASV and post-clustered ASV richness between platforms, suggesting that additional denoising step in the ASV workflow had effectively removed the ‘noisy’ reads. Both OTU-based and ASV-based composition were strongly correlated between the sequencing platforms, with essentially interchangeable results. Therefore, we conclude that DNBSEQ-G400RS and NovaSeq 6000 are both equally efficient high-throughput sequencing platforms to be utilized in studies aiming to apply the metabarcoding approach, but the main benefit of the former is related to lower sequencing cost.

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Hi-C chromosome conformation capture sequencing of avian genomes using the BGISEQ-500 platform

GigaScience ◽

10.1093/gigascience/giaa087 ◽

2020 ◽

Vol 9 (8) ◽

Author(s):

Marcela Sandoval-Velasco ◽

Juan Antonio Rodríguez ◽

Cynthia Perez Estrada ◽

Guojie Zhang ◽

Erez Lieberman Aiden ◽

...

Keyword(s):

Next Generation Sequencing ◽

High Throughput Sequencing ◽

Data Generation ◽

Next Generation ◽

Sequencing Data ◽

Yield Data ◽

Chromosome Conformation ◽

Sequencing Platform ◽

Sequencing Platforms ◽

Generation Sequencing

Abstract Background Hi-C experiments couple DNA-DNA proximity with next-generation sequencing to yield an unbiased description of genome-wide interactions. Previous methods describing Hi-C experiments have focused on the industry-standard Illumina sequencing. With new next-generation sequencing platforms such as BGISEQ-500 becoming more widely available, protocol adaptations to fit platform-specific requirements are useful to give increased choice to researchers who routinely generate sequencing data. Results We describe an in situ Hi-C protocol adapted to be compatible with the BGISEQ-500 high-throughput sequencing platform. Using zebra finch (Taeniopygia guttata) as a biological sample, we demonstrate how Hi-C libraries can be constructed to generate informative data using the BGISEQ-500 platform, following circularization and DNA nanoball generation. Our protocol is a modification of an Illumina-compatible method, based around blunt-end ligations in library construction, using un-barcoded, distally overhanging double-stranded adapters, followed by amplification using indexed primers. The resulting libraries are ready for circularization and subsequent sequencing on the BGISEQ series of platforms and yield data similar to what can be expected using Illumina-compatible approaches. Conclusions Our straightforward modification to an Illumina-compatible in situHi-C protocol enables data generation on the BGISEQ series of platforms, thus expanding the options available for researchers who wish to utilize the powerful Hi-C techniques in their research.

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re-Searcher: GUI-based bioinformatics tool for simplified genomics data mining of VCF files

PeerJ ◽

10.7717/peerj.11333 ◽

2021 ◽

Vol 9 ◽

pp. e11333

Author(s):

Daniyar Karabayev ◽

Askhat Molkenov ◽

Kaiyrgali Yerulanuly ◽

Ilyas Kabimoldayev ◽

Asset Daniyarov ◽

...

Keyword(s):

Web Application ◽

High Throughput Sequencing ◽

Sequencing Data ◽

Data Types ◽

Standard Format ◽

Standard Data ◽

Additional Information ◽

Link Type ◽

Sequencing Platforms ◽

User Friendly

Background High-throughput sequencing platforms generate a massive amount of high-dimensional genomic datasets that are available for analysis. Modern and user-friendly bioinformatics tools for analysis and interpretation of genomics data becomes essential during the analysis of sequencing data. Different standard data types and file formats have been developed to store and analyze sequence and genomics data. Variant Call Format (VCF) is the most widespread genomics file type and standard format containing genomic information and variants of sequenced samples. Results Existing tools for processing VCF files don’t usually have an intuitive graphical interface, but instead have just a command-line interface that may be challenging to use for the broader biomedical community interested in genomics data analysis. re-Searcher solves this problem by pre-processing VCF files by chunks to not load RAM of computer. The tool can be used as standalone user-friendly multiplatform GUI application as well as web application (https://nla-lbsb.nu.edu.kz). The software including source code as well as tested VCF files and additional information are publicly available on the GitHub repository (https://github.com/LabBandSB/re-Searcher).

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Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1601782113 ◽

2016 ◽

Vol 113 (19) ◽

pp. 5233-5238 ◽

Cited By ~ 79

Author(s):

Carl W. Fuller ◽

Shiv Kumar ◽

Mintu Porel ◽

Minchen Chien ◽

Arek Bibillo ◽

...

Keyword(s):

Dna Sequencing ◽

Real Time ◽

Lipid Bilayers ◽

Dna Polymerase ◽

Single Molecule ◽

High Throughput Sequencing ◽

Electrical Current ◽

Sequencing Data ◽

Sequencing By Synthesis ◽

Dna Strand

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

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An Extensive Meta-Metagenomic Search Identifies SARS-CoV-2-Homologous Sequences in Pangolin Lung Viromes

mSphere ◽

10.1128/msphere.00160-20 ◽

2020 ◽

Vol 5 (3) ◽

Cited By ~ 9

Author(s):

Lamia Wahba ◽

Nimit Jain ◽

Andrew Z. Fire ◽

Massa J. Shoura ◽

Karen L. Artiles ◽

...

Keyword(s):

Nucleic Acid ◽

High Speed ◽

High Throughput Sequencing ◽

Biological Significance ◽

Metagenomic Data ◽

Data Sets ◽

Sequencing Data ◽

Data Set ◽

Link Type ◽

Recent Emergence

ABSTRACT In numerous instances, tracking the biological significance of a nucleic acid sequence can be augmented through the identification of environmental niches in which the sequence of interest is present. Many metagenomic data sets are now available, with deep sequencing of samples from diverse biological niches. While any individual metagenomic data set can be readily queried using web-based tools, meta-searches through all such data sets are less accessible. In this brief communication, we demonstrate such a meta-metagenomic approach, examining close matches to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in all high-throughput sequencing data sets in the NCBI Sequence Read Archive accessible with the “virome” keyword. In addition to the homology to bat coronaviruses observed in descriptions of the SARS-CoV-2 sequence (F. Wu, S. Zhao, B. Yu, Y. M. Chen, et al., Nature 579:265–269, 2020, https://doi.org/10.1038/s41586-020-2008-3; P. Zhou, X. L. Yang, X. G. Wang, B. Hu, et al., Nature 579:270–273, 2020, https://doi.org/10.1038/s41586-020-2012-7), we note a strong homology to numerous sequence reads in metavirome data sets generated from the lungs of deceased pangolins reported by Liu et al. (P. Liu, W. Chen, and J. P. Chen, Viruses 11:979, 2019, https://doi.org/10.3390/v11110979). While analysis of these reads indicates the presence of a similar viral sequence in pangolin lung, the similarity is not sufficient to either confirm or rule out a role for pangolins as an intermediate host in the recent emergence of SARS-CoV-2. In addition to the implications for SARS-CoV-2 emergence, this study illustrates the utility and limitations of meta-metagenomic search tools in effective and rapid characterization of potentially significant nucleic acid sequences. IMPORTANCE Meta-metagenomic searches allow for high-speed, low-cost identification of potentially significant biological niches for sequences of interest.

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IROme, a New High-Throughput Molecular Tool for the Diagnosis of Inherited Retinal Dystrophies

BioMed Research International ◽

10.1155/2013/198089 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 14

Author(s):

Daniel F. Schorderet ◽

Alexandra Iouranova ◽

Tatiana Favez ◽

Leila Tiab ◽

Pascal Escher

Keyword(s):

Retinitis Pigmentosa ◽

Molecular Diagnosis ◽

High Throughput ◽

High Throughput Sequencing ◽

Sequence Variant ◽

Sequence Variants ◽

Sequencing Platform ◽

Capture Assay ◽

Retinal Dystrophies ◽

Exon Capture

The molecular diagnosis of retinal dystrophies is difficult because of the very important number of genes implicated and is rarely helped by genotype-phenotype correlations. This prompted us to develop IROme, a custom designed in solution-based targeted exon capture assay (SeqCap EZ Choice library, Roche NimbleGen) for 60 retinitis pigmentosa-linked genes and three candidate genes (942 exons). Pyrosequencing was performed on a Roche 454 GS Junior benchtop high-throughput sequencing platform. In total, 23 patients affected by retinitis pigmentosa were analyzed. Per patient, 39.6 Mb were generated, and 1111 sequence variants were detected on average, at a median coverage of 17-fold. After data filtering and sequence variant prioritization, disease-causing mutations were identified inABCA4,CNGB1,GUCY2D,PROM1,PRPF8,PRPF31,PRPH2,RHO,RP2, andTULP1for twelve patients (55%), ten mutations having never been reported previously. Potential mutations were identified in 5 additional patients, and in only 6 patients no molecular diagnosis could be established (26%). In conclusion, targeted exon capture and next-generation sequencing are a valuable and efficient approach to identify disease-causing sequence variants in retinal dystrophies.

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Comparative performance of the GenoLab M and NovaSeq 6000 sequencing platforms for transcriptome and LncRNA analysis

BMC Genomics ◽

10.1186/s12864-021-08150-8 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Yongfeng Liu ◽

Ran Han ◽

Letian Zhou ◽

Mingjie Luo ◽

Lidong Zeng ◽

...

Keyword(s):

Next Generation Sequencing ◽

High Performance ◽

Next Generation ◽

Sequencing Data ◽

Comparative Performance ◽

Sequencing Platform ◽

Next Generation Sequencing Platform ◽

Alternatively Spliced ◽

Sequencing Platforms ◽

Generation Sequencing

Abstract Background GenoLab M is a recently established next-generation sequencing platform from GeneMind Biosciences. Presently, Illumina sequencers are the globally leading sequencing platform in the next-generation sequencing market. Here, we present the first report to compare the transcriptome and LncRNA sequencing data of the GenoLab M sequencer to NovaSeq 6000 platform in various types of analysis. Results We tested 16 libraries in three species using various library kits from different companies. We compared the data quality, genes expression, alternatively spliced (AS) events, single nucleotide polymorphism (SNP), and insertions–deletions (InDel) between two sequencing platforms. The data suggested that platforms have comparable sensitivity and accuracy in terms of quantification of gene expression levels with technical compatibility. Conclusions Genolab M is a promising next-generation sequencing platform for transcriptomics and LncRNA studies with high performance at low costs.

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A workflow for accurate metabarcoding using nanopore MinION sequencing

10.1101/2020.05.21.108852 ◽

2020 ◽

Cited By ~ 2

Author(s):

Bilgenur Baloğlu ◽

Zhewei Chen ◽

Vasco Elbrecht ◽

Thomas Braukmann ◽

Shanna MacDonald ◽

...

Keyword(s):

High Throughput Sequencing ◽

Sequence Data ◽

Rolling Circle Amplification ◽

Error Rates ◽

Read Length ◽

Taxonomic Assignment ◽

Major Drawback ◽

Rolling Circle ◽

Sequencing Platform ◽

Sequencing Platforms

AbstractMetabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short-read high-throughput sequencing platforms. The Oxford Nanopore MinION™, a portable sequencing platform, represents a low-cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%.To test if the MinION™ represents a viable alternative to other sequencing platforms we used rolling circle amplification (RCA) to generate full-length consensus DNA barcodes (658bp of cytochrome oxidase I - COI) for a bulk mock sample of 50 aquatic invertebrate species. By applying two different laboratory protocols, we generated two MinION™ runs that were used to build consensus sequences. We also developed a novel Python pipeline, ASHURE, for processing, consensus building, clustering, and taxonomic assignment of the resulting reads.We were able to show that it is possible to reduce error rates to a median accuracy of up to 99.3% for long RCA fragments (>45 barcodes). Our pipeline successfully identified all 50 species in the mock community and exhibited comparable sensitivity and accuracy to MiSeq. The use of RCA was integral for increasing consensus accuracy, but it was also the most time-consuming step during the laboratory workflow and most RCA reads were skewed towards a shorter read length range with a median RCA fragment length of up to 1262bp. Our study demonstrates that Nanopore sequencing can be used for metabarcoding but we recommend the exploration of other isothermal amplification procedures to improve consensus length.

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Access COI barcode efficiently using high throughput Single-End 400 bp sequencing

10.1101/498618 ◽

2018 ◽

Cited By ~ 1

Author(s):

Chentao Yang ◽

Shangjin Tan ◽

Guangliang Meng ◽

David G. Bourne ◽

Paul A. O’Brien ◽

...

Keyword(s):

Dna Barcoding ◽

High Throughput ◽

High Throughput Sequencing ◽

Rapid Development ◽

Full Length ◽

List Type ◽

Sequencing Data ◽

Test Plate ◽

Function Modules ◽

Sequencing Platforms

SummaryOver the last decade, the rapid development of high-throughput sequencing platforms has accelerated species description and assisted morphological classification through DNA barcoding. However, constraints in barcoding costs led to unbalanced efforts which prevented accurate taxonomic identification for biodiversity studies.We present a high throughput sequencing approach based on the HIFI-SE pipeline which takes advantage of Single-End 400 bp (SE400) sequencing data generated by BGISEQ-500 to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons. HIFI-SE was written in Python and included four function modules of filter, assign, assembly and taxonomy.We applied the HIFI-SE to a test plate which contained 96 samples (30 corals, 64 insects and 2 blank controls) and delivered a total of 86 fully assembled HIFI COI barcodes. By comparing to their corresponding Sanger sequences (72 sequences available), it showed that most of the samples (98.61%, 71/72) were correctly and accurately assembled, including 46 samples that had a similarity of 100% and 25 of ca. 99%.Our approach can produce standard full-length barcodes cost efficiently, allowing DNA barcoding for global biomes which will advance DNA-based species identification for various ecosystems and improve quarantine biosecurity efforts.

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Accuracy and Reproducibility of Somatic Point Mutation Calling in Clinical-Type Targeted Sequencing Data

10.1101/2019.12.31.891952 ◽

2019 ◽

Author(s):

Ali Karimnezhad ◽

Gareth A. Palidwor ◽

Kednapa Thavorn ◽

David J. Stewart ◽

Pearl A. Campbell ◽

...

Keyword(s):

False Positive ◽

High Throughput Sequencing ◽

Variant Calling ◽

High Sensitivity ◽

Ground Truth ◽

Targeted Sequencing ◽

Sequencing Data ◽

Sequencing Platform ◽

Ion Torrent Pgm ◽

Fresh Frozen

AbstractBackgroundTreating cancer depends in part on identifying the mutations driving each patient’s disease. Many clinical laboratories are adopting high-throughput sequencing for assaying patients’ tumours, applying targeted panels to formalin-fixed paraffin-embedded tumour tissues to detect clinically-relevant mutations. While there have been some benchmarking and best practices studies of this scenario, much variant-calling work focuses on whole-genome or whole-exome studies, with fresh or fresh-frozen tissue. Thus, definitive guidance on best choices for sequencing platforms, sequencing strategies, and variant calling for clinical variant detection is still being developed.ResultsBecause ground truth for clinical specimens is rarely known, we used the well-characterized Coriell cell lines GM12878 and GM12877 to generate data. We prepared samples to mimic as closely as possible clinical biopsies, including formalin fixation and paraffin embedding. We evaluated two well-known targeted sequencing panels, Illumina’s TruSight 170 panel and the Oncomine Focus panel. Sequencing was performed on an Illumina NextSeq500 and an Ion Torrent PGM respectively. We performed multiple biological replicates of each assay, to test reproducibility. Finally, we applied five different public and freely-available somatic single-nucleotide variant (SNV) callers to the data, MuTect2, SAMtools, VarScan2, Pisces and VarDict. Although the TruSight 170 and Oncomine Focus panels cover different amounts of the genome, we did not observe major differences in variant calling success within the regions that each covers. We observed substantial discrepancies between the five variant callers. All had high sensitivity, detecting known SNVs, but highly varying and non-overlapping false positive detections. Harmonizing variant caller parameters or intersecting the results of multiple variant callers reduced disagreements. However, intersecting results from biological replicates was even better at eliminating false positives.ConclusionsReproducibility and accuracy of targeted clinical sequencing results depends less on sequencing platform and panel than on downstream bioinformatics and biological variability. Differences in variant callers’ default parameters are a greater influence on algorithm disagreement than other differences between the algorithms. Contrary to typical clinical practice, we recommend analyzing replicate samples, as this greatly decreases false positive calls.

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Great differences in performance and outcome of high-throughput sequencing data analysis platforms for fungal metabarcoding

10.7287/peerj.preprints.27019v2 ◽

2018 ◽

Author(s):

Sten Anslan ◽

Henrik Nilsson ◽

Christian Wurzbacher ◽

Petr Baldrian ◽

Leho Tedersoo ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Computation Time ◽

Potential Effect ◽

Data Sets ◽

Sequencing Data ◽

Data Set ◽

Operational Taxonomic Units ◽

High Throughput Sequencing Data ◽

Recent Developments

Along with recent developments in high-throughput sequencing (HTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appear to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon data set. We conclude that the output of each platform require manual validation of the OTUs by examining the taxonomy assignment values.

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