Amplicon Library Preparation v1 (protocols.io.bmuck6sw)

protocols.io ◽  
2020 ◽  
Author(s):  
Ariel Rabines ◽  
Rob Lampe ◽  
Andrew Allen
Keyword(s):  
Library Preparation ◽  
Amplicon Library

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

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

Ultradeep-Amplicon Pyrosequencing for Mutation Detection in the Kinase Domain of BCR-ABL Revealed Artificial Low-Level Variants That Need to Be Avoided for Relevant Mutational Data Interpretation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1396-1396
Author(s):  
Adela Brouckova ◽  
Simona Soverini ◽  
Caterina De Benedittis ◽  
Hana Klamova ◽  
Cyril Salek ◽  
...  
Keyword(s):  
Kinase Domain ◽  
Library Preparation ◽  
Nucleotide Substitutions ◽  
Taq Polymerase ◽  
Low Level ◽  
Amplicon Library ◽  
Proof Reading ◽  
Amplicon Pyrosequencing ◽  
Tki Treatment

Abstract Abstract 1396 Introduction. Mutations in the kinase domain (KD) of BCR-ABL are a known mechanism of resistance to the tyrosine kinase inhibitor (TKI) treatment of patients with chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). The gold standard for the routine examination of mutations in patients with therapy failure is the Sanger sequencing of amplified cDNA region of BCR-ABL KD with the sensitivity of ∼15–20%. More than 100 BCR-ABL KD mutations were described in association with development of resistance to imatinib. It was suggested that early ultra-sensitive detection of KD BCR-ABL mutations, at the time of diagnosis or shortly after imatinib start and before switch to 2nd generation TKI, may predict their subsequent expansion and failure of the treatment if the patient is treated with TKI against which is the type of mutation resistant. Ultradeep amplicon pyrosequencing represents a promising tool to study BCR-ABL mutation development and their expansion under TKI treatment. In our study, the detection of mutations reached the sensitivity of 0.03-0.01%. At this level we may expect detection of artifacts introduced during amplicon library preparation and pyrosequencing. Aims. The aim is to define the threshold for relevant detection of published BCR-ABL mutations (n= 124). Methods. We evaluated 155 CML or Ph+ALL patient samples. As the non-mutated control, the cDNA of KD c-ABL of healthy individuals (n= 9) and cDNA of wild type BCR-ABL from Ph+ cell lines (n=3) were used. To cover cDNA region of kinase domain (950 bp), multiple amplicons with the length up to 400 bp should be prepared to apply 454 technology. The experiments consist of: 1) comparison of two amplicon designs with 3 and 4 fragments covering KD to follow the amplicon-related artifacts; 2) the reproducibility test of 5 samples using 3 and 4 PCR fragments for amplicon library preparation; 3) tests with 2 different reverse transcriptases and Taq polymerases to detect artifactual nucleotide substitutions. Results. Using 454 technology we detected high number of ultralow-level single nucleotide substitutions (∼ 200) with the frequency < 1 in 100 in all samples analyzed including non-mutated controls using 3 and also 4 amplicon design. Surprisingly, we found that almost all low-level variants were T#x2610;#x0025;C or A#x2610;#x0025;G substitutions (Fig. 1), including those that may result in clinically relevant mutations (V288A, M244V, V289A, F317L, F359L, M351T, E355G, E453G). Evaluation of pyrograms suggested that they were unlikely to be errors of pyrosequencing, because they were not necessarily associated with hompolymer regions. The frequency of T#x2610;#x0025;C or A#x2610;#x0025;G low-level variants was lower when using proof-reading Taq polymerase. The pattern of these nucleotide substitutions is similar to that reported in IGH hypermutation analysis in CLL (Campbell et al. PNAS 2008) showing that T#x2610;#x0025;C or A#x2610;#x0025;G substitutions are errors caused by Taq polymerases. This is supported by the studies on PCR fidelity suggesting that Taq polymerase caused this pattern of base substitution even when using a proof-reading enzyme. Thus, the T#x2610;#x0025;C or A#x2610;#x0025;G substitution in BCR-ABL KD should not be evaluated below the level of 1%. The other but less frequent transitions caused by Taq polymerase are G#x2610;#x0025;A or C#x2610;#x0025;T substitutions that were detected in our study with the frequency < 1 in 1000, thus, the variants detected below 0.1% should not be examined as the true-positive result. Conclusion. Mutation data from ultradeep amplicon sequencing of BCR-ABL KD below 1% must be evaluated cautiously to avoid misleading interpretations. Attention must be paid to the fact that T#x2610;#x0025;C or A#x2610;#x0025;G substitution may arise spontaneously in vivo, but there is no way to differentiate in vivo spontaneous mutations from Taq polymerase errors in vitro. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy. Machova Polakova:Novartis: Honoraria, Research Funding; BMS: Honoraria.

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

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 A broadly applicable COI primer pair and an efficient single‐tube amplicon library preparation protocol for metabarcoding

2018 ◽  
Vol 8 (24) ◽  
pp. 12335-12350 ◽  
Author(s):  
Oskar Rennstam Rubbmark ◽  
Daniela Sint ◽  
Nina Horngacher ◽  
Michael Traugott
Keyword(s):  
Library Preparation ◽  
Single Tube ◽  
Amplicon Library ◽  
Library Preparation Protocol

scholarly journals 423. SARS-CoV-2 NGS Assay Powered by Biotia COVID-DX Software

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S278-S279
Author(s):  
Dorottya Nagy-Szakal ◽  
Mara Couto-Rodriguez ◽  
Joseph Barrows ◽  
Heather L Wells ◽  
Marilyne Debieu ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Viral Genome ◽  
Board Member ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Analysis Tool ◽  
Viral Titer ◽  
Target Enrichment ◽  
Library Preparation ◽  
Software Analysis

Abstract Background COVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome. Methods The SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis. Results The SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided &gt;99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens. Conclusion The SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease. Disclosures Dorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

scholarly journals A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathy E. Raven ◽  
Sophia T. Girgis ◽  
Asha Akram ◽  
Beth Blane ◽  
Danielle Leek ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Clinical Microbiology ◽  
Bacterial Species ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
Simultaneous Processing ◽  
Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

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