scholarly journals Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

2019 ◽  
Author(s):  
Rachel L. Marine ◽  
Laura C. Magaña ◽  
Christina J. Castro ◽  
Kun Zhao ◽  
Anna M. Montmayeur ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
Ion Torrent ◽  
Library Preparation ◽  
Genome Sequences ◽  
Sequencing Platform ◽  
Viral Genomes ◽  
Ion Torrent Pgm ◽  
Miseq Platform ◽  
Sequencing Platforms ◽  
The Cost

ABSTRACTNext-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. We evaluated the Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 outperformed the older Ion Torrent PGM platform in data quality and cost, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 or Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 or Illumina MiSeq V2) the cost per sample was comparable. These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.

scholarly journals Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2093
Author(s):  
Shen-Yuan Hsieh ◽  
Mohammad A. Tariq ◽  
Andrea Telatin ◽  
Rebecca Ansorge ◽  
Evelien M. Adriaenssens ◽  
...  
Keyword(s):  
Alpha Diversity ◽  
Diversity Indices ◽  
P Value ◽  
Library Preparation ◽  
High Genetic Diversity ◽  
Sequencing Platform ◽  
Viral Genomes ◽  
Healthy Donors ◽  
Faecal Samples ◽  
Sequencing Platforms

The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as “viral dark matter”. However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating “rare” members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses.

cpn60 metagenomic amplicon library preparation for the Illumina Miseq platform

2021 ◽  
Author(s):  
Champika Fernando ◽  
Janet E. Hill
Keyword(s):  
Dna Sequences ◽  
Illumina Miseq ◽  
Library Preparation ◽  
Illumina Miseq Platform ◽  
Wide Range ◽  
Miseq Platform ◽  
Identification Of Bacteria ◽  
Sequencing Platforms ◽  
Almost All ◽  
Level Identification

Abstract This protocol can be applied to determine the composition of a microbial community. The cpn60 gene (also known as groEL, hsp60) is present in almost all bacteria and a 552-558 bp region of the gene has been established as a barcode for species level identification of bacteria. The primer cocktail used in this protocol amplifies cpn60 barcode sequences from bacteria with a wide range of G+C content. Some species of Mycoplasma lack the cpn60 gene and therefore this method is not recommended to detect Mycoplasma. DNA sequences generated from this method could be compared to cpnDB, a public database of cpn60 sequences, for identification. Library preparation involves cpn60 amplicon generation, PCR clean-up, index PCR, index PCR clean-up, library quantification, normalization, pooling, library denaturation and loading. Time taken to complete depends on the number of samples included. If using 96 samples, the procedure takes 8 hours but there are several stages where the samples could be stored and continued the next day. Specific instructions are provided for the Illumina MiSeq platform, but the protocol could easily be adapted for other sequencing platforms.

scholarly journals Efficient and stable metabarcoding sequencing from DNBSEQ-G400 sequencer examined by large fungal community analysis

2020 ◽  
Author(s):  
Xiaohuan Sun ◽  
Jingjing Wang ◽  
Chao Fang ◽  
Jiguang Li ◽  
Mo Han ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Large Scale ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Soil Samples ◽  
Its2 Region ◽  
Sequencing Platform ◽  
Sequencing Bias ◽  
Miseq Platform ◽  
Sequencing Platforms

ABSTRACTMetabarcoding has become the de facto method for characterizing the structure of microbial communities in complex environmental samples. To determine how sequencing platform may influence microbial community characterization, we present a large-scale comparison of two sequencing platforms; Illumina MiSeq and a new platform DNBSEQ-G400 developed by MGI Tech. The accuracy of DNBSEQ-G400 on bacterial and fungal mock samples and compared sequencing consistency and precision between DNBSEQ-G400 and MiSeq platforms by sequencing the fungal ITS2 region from 1144 soil samples with 3 technical replicates. The DNBSEQ-G400 showed a high accuracy in reproducing mock communities containing different proportions of bacteria and fungi, respectively. The taxonomic profiles of the 1144 soil samples generated by the two DNBSEQ-G400 modes closely resembled each other and were highly correlated with those generated by the MiSeq platform. Analyses of technical replicates demonstrated a run bias against certain taxa on the MiSeq but not DNBSEQ-G400 platform. Based on lower cost, greater capacity, and less bias, we conclude that DNBSEQ-G400 is an optimal platform for short-term metabarcoding of microbial communities.IMPORTANCEExperimental steps that generate sequencing bias during amplicon sequencing have been intensively evaluated, including the choice of primer pair, polymerase, PCR cycle and technical replication. However, few studies have assessed the accuracy and precision of different sequencing platforms. Here, we compared the performance of newly released DNBSEQ-G400 sequencer with that of the commonly used Illumina MiSeq platform by leveraging amplicon sequencing of a large number of soil samples. Significant sequencing bias among major fungal genera was found in parallel MiSeq runs, which can be easily neglected without the use of sequencing controls. We emphasize the importance of technical controls in large-scale sequencing efforts and provide DNBSEQ-G400 as an alternative with increased sequencing capacity and more stable reproducibility for amplicon sequencing.

scholarly journals Revealing the Complexities of Metabarcoding with a Diverse Arthropod Mock Community

2018 ◽  
Author(s):  
Thomas W. A. Braukmann ◽  
Natalia V. Ivanova ◽  
Sean W. J. Prosser ◽  
Vasco Elbrecht ◽  
Dirk Steinke ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
Ion Torrent ◽  
Mock Community ◽  
Species Recovery ◽  
Sequencing Platform ◽  
Ion Torrent Pgm ◽  
Terrestrial Arthropods ◽  
Dna Metabarcoding ◽  
Powerful Approach ◽  
Read Abundance

AbstractDNA 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.

scholarly journals Viral metagenomes of Lake Soyang, the largest freshwater lake in South Korea

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Kira Moon ◽  
Suhyun Kim ◽  
Ilnam Kang ◽  
Jang-Cheon Cho
Keyword(s):  
South Korea ◽  
Illumina Miseq ◽  
Freshwater Lakes ◽  
Genome Sequences ◽  
Illumina Miseq Platform ◽  
Viral Community ◽  
Viral Communities ◽  
Miseq Platform ◽  
Metagenome Sequencing ◽  
Freshwater Reservoir

Abstract A high number of viral metagenomes have revealed countless genomes of putative bacteriophages that have not yet been identified due to limitations in bacteriophage cultures. However, most virome studies have been focused on marine or gut environments, thereby leaving the viral community structure of freshwater lakes unclear. Because the lakes located around the globe have independent ecosystems with unique characteristics, viral community structures are also distinctive but comparable. Here, we present data on viral metagenomes that were seasonally collected at a depth of 1 m from Lake Soyang, the largest freshwater reservoir in South Korea. Through shotgun metagenome sequencing using the Illumina MiSeq platform, 3.08 to 5.54-Gbps of reads per virome were obtained. To predict the viral genome sequences within Lake Soyang, contigs were constructed and 648 to 1,004 putative viral contigs were obtained per sample. We expect that both viral metagenome reads and viral contigs would contribute in comparing and understanding of viral communities among different freshwater lakes depending on seasonal changes.

scholarly journals Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

2020 ◽  
Vol 280 ◽  
pp. 113865 ◽  
Author(s):  
Rachel L. Marine ◽  
Laura C. Magaña ◽  
Christina J. Castro ◽  
Kun Zhao ◽  
Anna M. Montmayeur ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Ion Torrent ◽  
Ion Torrent Pgm

scholarly journals Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation

2018 ◽  
Author(s):  
Madeline Y Mayday ◽  
Lillian M Khan ◽  
Eric D Chow ◽  
Matt S Zinter ◽  
Joseph L DeRisi
Keyword(s):  
Metagenomic Sequencing ◽  
Library Preparation ◽  
Sequencing Library ◽  
Reaction Volumes ◽  
Sequencing Platforms ◽  
Time Required ◽  
The Cost ◽  
Sequencing Library Preparation ◽  
Full Volume ◽  
Generation Sequencing

AbstractPreparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

scholarly journals Impact of DNA Sequencing and Analysis Methods on 16S rRNA Gene Bacterial Community Analysis of Dairy Products

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Zhengyao Xue ◽  
Mary E. Kable ◽  
Maria L. Marco
Keyword(s):  
16S Rrna ◽  
Dna Sequencing ◽  
Dairy Products ◽  
Bacterial Species ◽  
Ion Torrent ◽  
Mock Community ◽  
Sequencing Platform ◽  
Ion Torrent Pgm ◽  
Sequencing Method ◽  
Mock Communities

ABSTRACT DNA sequencing and analysis methods were compared for 16S rRNA V4 PCR amplicon and genomic DNA (gDNA) mock communities encompassing nine bacterial species commonly found in milk and dairy products. The two communities comprised strain-specific DNA that was pooled before (gDNA) or after (PCR amplicon) the PCR step. The communities were sequenced on the Illumina MiSeq and Ion Torrent PGM platforms and then analyzed using the QIIME 1 (UCLUST) and Divisive Amplicon Denoising Algorithm 2 (DADA2) analysis pipelines with taxonomic comparisons to the Greengenes and Ribosomal Database Project (RDP) databases. Examination of the PCR amplicon mock community with these methods resulted in operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) that ranged from 13 to 118 and were dependent on the DNA sequencing method and read assembly steps. The additional 4 to 109 OTUs/ASVs (from 9 OTUs/ASVs) included assignments to spurious taxa and sequence variants of the 9 species included in the mock community. Comparisons between the gDNA and PCR amplicon mock communities showed that combining gDNAs from the different strains prior to PCR resulted in up to 8.9-fold greater numbers of spurious OTUs/ASVs. However, the DNA sequencing method and paired-end read assembly steps conferred the largest effects on predictions of bacterial diversity, with effect sizes of 0.88 (Bray-Curtis) and 0.32 (weighted Unifrac), independent of the mock community type. Overall, DNA sequencing performed with the Ion Torrent PGM and analyzed with DADA2 and the Greengenes database resulted in the most accurate predictions of the mock community phylogeny, taxonomy, and diversity. IMPORTANCE Validated methods are urgently needed to improve DNA sequence-based assessments of complex bacterial communities. In this study, we used 16S rRNA PCR amplicon and gDNA mock community standards, consisting of nine, dairy-associated bacterial species, to evaluate the most commonly applied 16S rRNA marker gene DNA sequencing and analysis platforms used in evaluating dairy and other bacterial habitats. Our results show that bacterial metataxonomic assessments are largely dependent on the DNA sequencing platform and read curation method used. DADA2 improved sequence annotation compared with QIIME 1, and when combined with the Ion Torrent PGM DNA sequencing platform and the Greengenes database for taxonomic assignment, the most accurate representation of the dairy mock community standards was reached. This approach will be useful for validating sample collection and DNA extraction methods and ultimately investigating bacterial population dynamics in milk- and dairy-associated environments.

scholarly journals Draft Genome Sequences of Pseudoalteromonas telluritireducens DSM 16098 and P. spiralis DSM 16099 Isolated from the Hydrothermal Vents of the Juan de Fuca Ridge

2016 ◽  
Vol 4 (4) ◽  
Author(s):  
Huan Zhang ◽  
Rui Liu ◽  
Mengqiang Wang ◽  
Hao Wang ◽  
Qiang Gao ◽  
...  
Keyword(s):  
Hydrothermal Vents ◽  
Draft Genome ◽  
Juan De Fuca Ridge ◽  
Ion Torrent ◽  
Genome Sequences ◽  
Fuca Ridge ◽  
Ion Torrent Pgm ◽  
Juan De Fuca

This report describes the draft genome sequences of two strains, Pseudoalteromonas telluritireducens DSM 16098 and P. spiralis DSM 16099, which were isolated from hydrothermal vents of the Juan de Fuca Ridge. The reads generated by an Ion Torrent PGM were assembled into contigs with total sizes of 4.4 Mb and 4.1 Mb for DSM 16098 and DSM 16099, respectively.

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