High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ system for simplified amplicon library preparation

2013 ◽  
Vol 81 (3) ◽  
pp. 141-149 ◽  
Author(s):  
P. V. Moonsamy ◽  
T. Williams ◽  
P. Bonella ◽  
C. L. Holcomb ◽  
B. N. Höglund ◽  
...  
Keyword(s):  
High Throughput ◽  
454 Sequencing ◽  
Library Preparation ◽  
Amplicon Library ◽  
Hla Genotyping ◽  
Fluidigm Access Array

201-P Use of the Fluidigm® access Array™ system provides simplified amplicon library preparation in next generation sequencing for high throughput HLA genotyping

2011 ◽  
Vol 72 ◽  
pp. S142 ◽  
Author(s):  
Priscilla V. Moonsamy ◽  
Persia L. Bonella ◽  
Timothy C. Williams ◽  
Cherie L. Holcomb ◽  
Gregory S. Turenchalk ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Library Preparation ◽  
Next Generation ◽  
Amplicon Library ◽  
Hla Genotyping ◽  
Generation Sequencing

scholarly journals Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e96727 ◽  
Author(s):  
Ulrike Menzel ◽  
Victor Greiff ◽  
Tarik A. Khan ◽  
Ulrike Haessler ◽  
Ina Hellmann ◽  
...  
Keyword(s):  
High Throughput ◽  
Comprehensive Evaluation ◽  
Library Preparation ◽  
Preparation Methods ◽  
Amplicon Library

scholarly journals High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

mSystems ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Jeremiah J. Minich ◽  
Greg Humphrey ◽  
Rodolfo A. S. Benitez ◽  
Jon Sanders ◽  
Austin Swafford ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Ecology ◽  
High Throughput ◽  
Preparation Method ◽  
Library Preparation ◽  
Reaction Volume ◽  
Liquid Handling ◽  
Amplicon Library ◽  
The Cost ◽  
Library Preparation Method

ABSTRACT Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low-cost miniaturized (5-µl volume) high-throughput (384-sample) amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation to $1.42 per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5-µl reaction volume with 0.2 µl of genomic DNA (gDNA) and the standard 25-µl reaction volume with 1 µl of gDNA. Between the two methods, alpha diversity was highly correlated (R2 > 0.95), while distances of technical replicates were much lower than within-body-site variation (P < 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25-µl reactions to triplicate 5-µl reactions) are large enough to cover a MiSeq sequencing run for 768 samples while preserving accurate microbiome measurements. IMPORTANCE Reduced costs of sequencing have tremendously impacted the field of microbial ecology, allowing scientists to design more studies with larger sample sizes that often exceed 10,000 samples. Library preparation costs have not kept pace with sequencing prices, although automated liquid handling robots provide a unique opportunity to bridge this gap while also decreasing human error. Here, we take advantage of an acoustic liquid handling robot to develop a high-throughput miniaturized library preparation method of a highly cited and broadly used 16S rRNA gene amplicon reaction. We evaluate the potential negative effects of reducing the PCR volume along with varying the amount of gDNA going into the reaction. Our optimized method reduces sample-processing costs while continuing to generate a high-quality microbiome readout that is indistinguishable from the original method.

scholarly journals High-throughput miniaturized 16S rRNA amplicon library preparation v1 (protocols.io.u2ieyce)

protocols.io ◽  
2018 ◽  
Author(s):  
Jeremiah Minich ◽  
Greg Humphrey ◽  
Rodolfo Salido ◽  
Jon Sanders ◽  
Austin Swafford ◽  
...  
Keyword(s):  
16S Rrna ◽  
High Throughput ◽  
Library Preparation ◽  
Amplicon Library

scholarly journals Fast and Efficient 5′P Degradome Library Preparation for Analysis of Co-Translational Decay in Arabidopsis

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 466
Author(s):  
Marie-Christine Carpentier ◽  
Cécile Bousquet-Antonelli ◽  
Rémy Merret
Keyword(s):  
Gene Expression ◽  
Quality Control ◽  
Transcriptional Regulation ◽  
High Throughput ◽  
Library Preparation ◽  
Working Day ◽  
Set Up ◽  
Post Transcriptional Regulation ◽  
Commercial Kit

The recent development of high-throughput technologies based on RNA sequencing has allowed a better description of the role of post-transcriptional regulation in gene expression. In particular, the development of degradome approaches based on the capture of 5′monophosphate decay intermediates allows the discovery of a new decay pathway called co-translational mRNA decay. Thanks to these approaches, ribosome dynamics could now be revealed by analysis of 5′P reads accumulation. However, library preparation could be difficult to set-up for non-specialists. Here, we present a fast and efficient 5′P degradome library preparation for Arabidopsis samples. Our protocol was designed without commercial kit and gel purification and can be easily done in one working day. We demonstrated the robustness and the reproducibility of our protocol. Finally, we present the bioinformatic reads-outs necessary to assess library quality control.

scholarly journals Environmental Sequencing Provides Reasonable Estimates of the Relative Abundance of Specific Picoeukaryotes

2016 ◽  
Vol 82 (15) ◽  
pp. 4757-4766 ◽  
Author(s):  
Caterina R. Giner ◽  
Irene Forn ◽  
Sarah Romac ◽  
Ramiro Logares ◽  
Colomban de Vargas ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
High Throughput ◽  
Relative Abundance ◽  
Ribosomal Dna ◽  
18S Rdna ◽  
High Throughput Sequencing ◽  
454 Sequencing ◽  
Epifluorescence Microscopy ◽  
Environmental Sequencing

ABSTRACTHigh-throughput sequencing (HTS) is revolutionizing environmental surveys of microbial diversity in the three domains of life by providing detailed information on which taxa are present in microbial assemblages. However, it is still unclear how the relative abundance of specific taxa gathered by HTS correlates with cell abundances. Here, we quantified the relative cell abundance of 6 picoeukaryotic taxa in 13 planktonic samples from 6 European coastal sites using epifluorescence microscopy on tyramide signal amplification-fluorescencein situhybridization preparations. These relative abundance values were then compared with HTS data obtained in three separate molecular surveys: 454 sequencing of the V4 region of the 18S ribosomal DNA (rDNA) using DNA and RNA extracts (DNA-V4 and cDNA-V4) and Illumina sequencing of the V9 region (cDNA-V9). The microscopic and molecular signals were generally correlated, indicating that a relative increase in specific 18S rDNA was the result of a large proportion of cells in the given taxa. Despite these positive correlations, the slopes often deviated from 1, precluding a direct translation of sequences to cells. Our data highlighted clear differences depending on the nucleic acid template or the 18S rDNA region targeted. Thus, the molecular signal obtained using cDNA templates was always closer to relative cell abundances, while the V4 and V9 regions gave better results depending on the taxa. Our data support the quantitative use of HTS data but warn about considering it as a direct proxy of cell abundances.IMPORTANCEDirect studies on marine picoeukaryotes by epifluorescence microscopy are problematic due to the lack of morphological features and due to the limited number and poor resolution of specific phylogenetic probes used in fluorescencein situhybridization (FISH) routines. As a consequence, there is an increasing use of molecular methods, including high-throughput sequencing (HTS), to study marine microbial diversity. HTS can provide a detailed picture of the taxa present in a community and can reveal diversity not evident using other methods, but it is still unclear what the meaning of the sequence abundance in a given taxon is. Our aim is to investigate the correspondence between the relative HTS signal and relative cell abundances in selected picoeukaryotic taxa. Environmental sequencing provides reasonable estimates of the relative abundance of specific taxa. Better results are obtained when using RNA extracts as the templates, while the region of 18S ribosomal DNA had different influences depending on the taxa assayed.

scholarly journals Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 566 ◽  
Author(s):  
Siemon Ng ◽  
Cassandra Braxton ◽  
Marc Eloit ◽  
Szi Feng ◽  
Romain Fragnoud ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Nucleic Acids ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Virus Detection ◽  
Extraction Methods ◽  
Control Measures ◽  
Acid Extraction ◽  
Library Preparation ◽  
Sample Processing

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.

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

PLoS ONE ◽  
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.

scholarly journals Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

BMC Genomics ◽  
2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Wells W. Wu ◽  
Je-Nie Phue ◽  
Chun-Ting Lee ◽  
Changyi Lin ◽  
Lai Xu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Library Preparation ◽  
Next Generation ◽  
Generation Sequencing

High Throughput Semi-Automated SARS-CoV-2 Library Preparation Protocol for Ion Torrent Sequencing using Opentrons, New England Biolabs Kit, and ARTIC Primers v2

protocols.io ◽  
2021 ◽  
Author(s):  
Elias Dahdouh ◽  
Fernando Lázaro Perona ◽  
María Rodríguez Tejedor ◽  
Rubén Cáceres Sánchez ◽  
Iván Bloise Sánchez ◽  
...  
Keyword(s):  
New England ◽  
High Throughput ◽  
Ion Torrent ◽  
Library Preparation ◽  
England Biolabs ◽  
Ion Torrent Sequencing ◽  
Library Preparation Protocol
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