Application of a Sanger-Based External Quality Assurance Strategy for the Transition of HIV-1 Drug Resistance Assays to Next Generation Sequencing

The Nationwide Institute of Allergy and Infectious Ailments (NIAID) Virology High quality Assurance (VQA) established a sturdy proficiency testing program for Sanger sequencing (SS)-based HIV-1 drug resistance (HIVDR) testing in 2001. Whereas most of the classes discovered through the growth of such applications may additionally apply to subsequent technology sequencing (NGS)-based HIVDR assays, challenges stay for the continued analysis of NGS-based testing. These challenges embrace a correct evaluation of assay accuracy and the reproducibility of low abundance variant detection, intra- and inter-assay efficiency comparisons amongst laboratories utilizing lab-defined assessments, and totally different knowledge evaluation pipelines designed for NGS. In collaboration with the World Well being Group (WHO) International HIVDR Laboratory Community and the Public Well being Company of Canada, the Rush VQA program distributed archived proficiency testing panels to 10 laboratories to guage internally developed NGS assays.
Consensus FASTA information have been submitted utilizing 5%, 10%, and 20% variant detection thresholds, and scored based mostly on the identical standards used for SS. This small research confirmed that the SS Exterior High quality Assurance (EQA) strategy can be utilized as a transitional technique for utilizing NGS to generate SS-like knowledge and for ongoing efficiency whereas utilizing NGS knowledge from the identical high quality management supplies to additional consider NGS assay efficiency. Lung most cancers (LC) is the primary reason behind demise for most cancers worldwide and non-small cell lung most cancers (NSCLC) represents the commonest histology.
The invention of genomic alterations in driver genes that supply the opportunity of therapeutic intervention has fully modified the strategy to the analysis and remedy of superior NSCLC sufferers, and tumor molecular profiling has grow to be necessary for the selection of probably the most acceptable therapeutic technique. Nevertheless, in roughly 30% of NSCLC sufferers tumor tissue is insufficient for biomarker evaluation. The event of extremely delicate subsequent technology sequencing (NGS) applied sciences for the evaluation of circulating cell-free DNA (cfDNA) is rising as a invaluable different to evaluate tumor molecular panorama in case of tissue unavailability. Moreover, cfDNA NGS testing can higher recapitulate NSCLC heterogeneity as in contrast with tissue testing. On this overview we describe the primary benefits and limits of utilizing NGS-based cfDNA evaluation to information the therapeutic decision-making course of in superior NSCLC sufferers, to watch the response to remedy and to establish mechanisms of resistance early.

 

 

Fast EGFR Mutation Detection Utilizing the Idylla Platform: Single establishment expertise of 1200 instances analyzed by an in-house developed pipeline and comparability with concurrent subsequenttechnology sequencing outcomes

Mutations within the epidermal development issue receptor (EGFR) are the commonest targetable alterations in lung adenocarcinoma. To facilitate speedy testing, we integrated the Idylla EGFR assay as screening technique previous to subsequent technology sequencing (NGS). We describe our validation and expertise utilizing an in-house developed evaluation pipeline, enhanced with a guide overview algorithm. Outcomes are in contrast with corresponding NGS outcomes. In all, 1,249 samples have been studied. Validation demonstrated 98.57%  concordance with the reference strategies. The restrict of detection diverse from 2 to five% variant allele frequency if whole EGFR Cq was between 20 and 23. Of 1179 scientific instances, 23.41% have been EGFR constructive by Idylla.
Concurrent NGS was efficiently carried out on 94.9%  of requests. Concordance of Idylla with NGS was 98.62% and 98.50%  utilizing our in-house and Idylla evaluation pipelines, respectively. Discordances concerned missed mutations by each assays related to low tumor/low enter. Incorporating a guide overview algorithm to complement automated calls, improved accuracy from 98.62 to 99.37% and sensitivity from 94.68% to 97.58%. Total reporting time, from receipt of fabric to official scientific report, ranged from 1-Three days. We conclude that Idylla EGFR testing permits speedy and delicate screening with out compromising subsequent complete NGS, when required. Subsequent-generation sequencing (NGS) is more and more being adopted as a invaluable technique for the detection of somatic variants in scientific oncology.
Nevertheless, it’s nonetheless difficult to succeed in a passable degree of robustness and standardization in scientific follow when utilizing the at the moment obtainable bioinformatics pipelines to detect variants from uncooked sequencing knowledge. Furthermore, acceptable reference datasets are missing for scientific bioinformatics pipeline growth, validation and proficiency testing. Right here, we developed VarBen, an open-source software program for variant simulation to generate custom-made reference datasets by straight enhancing the unique sequencing reads. VarBen can introduce quite a lot of variants, together with single-nucleotide variants, small insertions and deletions, and enormous structural variants, into focused, exome or whole-genome sequencing knowledge, and might deal with sequencing knowledge from each the Illumina and Ion Torrent sequencing platforms.
Application of a Sanger-Based External Quality Assurance Strategy for the Transition of HIV-1 Drug Resistance Assays to Next Generation Sequencing
Application of a Sanger-Based External Quality Assurance Strategy for the Transition of HIV-1 Drug Resistance Assays to Next Generation Sequencing

Analysis of Ion Torrent subsequenttechnology sequencing for thalassemia analysis

Introduction: To guage a next-generation sequencing (NGS) workflow within the screening and analysis of thalassemia.
StrategiesOn this potential research, blood samples have been obtained from folks present process genetic screening for thalassemia at our centre in Guangzhou, China. Genomic DNA was polymerase chain response (PCR)-amplified and sequenced utilizing the Ion Torrent system and outcomes in contrast with conventional genetic analyses.
OutcomesOf the 359 topics, 148 (41%) have been confirmed to have thalassemia. Variant detection recognized 35 differing types together with the commonest. Identification of the mutational websites by NGS have been in keeping with these recognized by Sanger sequencing and Hole-PCR. The sensitivity and specificities of the Ion Torrent NGS have been 100%. In a separate take a look at of 16 samples, outcomes have been constant when repeated ten instances.

Chymotrypsin for Sequencing grade

C4001-100 100ug
EUR 286

Trypin for Mass & Sequencing

T9600-025 25ug
EUR 145

Trypin for Mass & Sequencing

T9600-100 100ug
EUR 219

Trypin for Mass & Sequencing

T9600-112 12x100ug
EUR 1657

Trypin for Mass & Sequencing

T9600-400 4x100ug
EUR 651

SequaGel Sequencing System 1L Kit

NAT1136 1KIT
EUR 143

SequaGel Sequencing System 2.2L Kit

NAT1138 EACH
EUR 211

PCR Clean Up for DNA Sequencing

BT5100 100preps
EUR 95.68

PCR Clean Up for DNA Sequencing

BT5101 1000Preps, 1000prep
EUR 461.08

DNA Library Prep Kit for IIlumina Sequencing

K1475-12 12 Rxns
EUR 480

Random Primers

S300 30 µg
EUR 46

Random Primers

S305 5x30 µg
EUR 102

U1 Primers

MP00001 150 ul / 10 uM
EUR 121

SNORD44 Primers

MPH00003 150 ul / 10 uM
EUR 121

SNORD47 Primers

MPH00004 150 ul / 10 uM
EUR 121

SNORD48 Primers

MPH00005 150 ul / 10 uM
EUR 121

RNU43 Primers

MPM00003 150 ul / 10 uM
EUR 121

snoRNA142 Primers

MPM00004 150 ul / 10 uM
EUR 121

U6-2 Primers

MPH00001 150 ul / 10 uM
EUR 121

U6 snRNA Primers

MPM00002 150 ul / 10 uM
EUR 121

U1 snRNA Primers

MPM00006 150 ul / 10 uM
EUR 121

rno-miR-323 Primers

MP-r00001 150 ul / 10 uM
EUR 176

rno-miR-301a Primers

MP-r00002 150 ul / 10 uM
EUR 176

rno-miR-326 Primers

MP-r00005 150 ul / 10 uM
EUR 176

rno-miR-327 Primers

MP-r00006 150 ul / 10 uM
EUR 176

rno-let-7d Primers

MP-r00007 150 ul / 10 uM
EUR 176

rno-miR-328 Primers

MP-r00008 150 ul / 10 uM
EUR 176

rno-miR-329 Primers

MP-r00009 150 ul / 10 uM
EUR 176

rno-miR-331 Primers

MP-r00010 150 ul / 10 uM
EUR 176

rno-miR-140 Primers

MP-r00011 150 ul / 10 uM
EUR 176

rno-miR-335 Primers

MP-r00012 150 ul / 10 uM
EUR 176

rno-miR-336 Primers

MP-r00013 150 ul / 10 uM
EUR 176

rno-miR-337 Primers

MP-r00014 150 ul / 10 uM
EUR 176

rno-miR-338 Primers

MP-r00015 150 ul / 10 uM
EUR 176

rno-miR-341 Primers

MP-r00017 150 ul / 10 uM
EUR 176

rno-miR-343 Primers

MP-r00018 150 ul / 10 uM
EUR 176

rno-miR-346 Primers

MP-r00020 150 ul / 10 uM
EUR 176

rno-miR-347 Primers

MP-r00021 150 ul / 10 uM
EUR 176

rno-miR-349 Primers

MP-r00022 150 ul / 10 uM
EUR 176

rno-miR-129 Primers

MP-r00023 150 ul / 10 uM
EUR 176

rno-miR-20a Primers

MP-r00024 150 ul / 10 uM
EUR 176

rno-miR-350 Primers

MP-r00025 150 ul / 10 uM
EUR 176

rno-miR-7a Primers

MP-r00026 150 ul / 10 uM
EUR 176

rno-miR-7a Primers

MP-r00027 150 ul / 10 uM
EUR 176

rno-miR-351 Primers

MP-r00028 150 ul / 10 uM
EUR 176

rno-miR-352 Primers

MP-r00029 150 ul / 10 uM
EUR 176

rno-miR-135b Primers

MP-r00030 150 ul / 10 uM
EUR 176

rno-miR-151 Primers

MP-r00031 150 ul / 10 uM
EUR 176

rno-miR-101b Primers

MP-r00032 150 ul / 10 uM
EUR 176

rno-let-7a Primers

MP-r00033 150 ul / 10 uM
EUR 176

rno-let-7b Primers

MP-r00034 150 ul / 10 uM
EUR 176

rno-let-7c Primers

MP-r00035 150 ul / 10 uM
EUR 176

rno-let-7e Primers

MP-r00036 150 ul / 10 uM
EUR 176

rno-let-7f Primers

MP-r00037 150 ul / 10 uM
EUR 176

rno-let-7i Primers

MP-r00038 150 ul / 10 uM
EUR 176

rno-miR-7b Primers

MP-r00039 150 ul / 10 uM
EUR 176

rno-miR-9 Primers

MP-r00040 150 ul / 10 uM
EUR 176

rno-miR-10b Primers

MP-r00042 150 ul / 10 uM
EUR 176

rno-miR-15b Primers

MP-r00043 150 ul / 10 uM
EUR 176

rno-miR-16 Primers

MP-r00044 150 ul / 10 uM
EUR 176

rno-miR-18a Primers

MP-r00047 150 ul / 10 uM
EUR 176

rno-miR-19b Primers

MP-r00048 150 ul / 10 uM
EUR 176

rno-miR-19a Primers

MP-r00049 150 ul / 10 uM
EUR 176

rno-miR-21 Primers

MP-r00050 150 ul / 10 uM
EUR 176

rno-miR-22 Primers

MP-r00051 150 ul / 10 uM
EUR 176

rno-miR-23a Primers

MP-r00052 150 ul / 10 uM
EUR 176

rno-miR-23b Primers

MP-r00053 150 ul / 10 uM
EUR 176

rno-miR-24 Primers

MP-r00054 150 ul / 10 uM
EUR 176

rno-miR-24 Primers

MP-r00055 150 ul / 10 uM
EUR 176

rno-miR-25 Primers

MP-r00056 150 ul / 10 uM
EUR 176

rno-miR-26a Primers

MP-r00057 150 ul / 10 uM
EUR 176

rno-miR-26b Primers

MP-r00058 150 ul / 10 uM
EUR 176

rno-miR-27b Primers

MP-r00059 150 ul / 10 uM
EUR 176

rno-miR-27a Primers

MP-r00060 150 ul / 10 uM
EUR 176

rno-miR-28 Primers

MP-r00061 150 ul / 10 uM
EUR 176

rno-miR-29b Primers

MP-r00062 150 ul / 10 uM
EUR 176

rno-miR-29b Primers

MP-r00063 150 ul / 10 uM
EUR 176

rno-miR-29a Primers

MP-r00064 150 ul / 10 uM
EUR 176

rno-miR-29c Primers

MP-r00065 150 ul / 10 uM
EUR 176

rno-miR-30c Primers

MP-r00066 150 ul / 10 uM
EUR 176

rno-miR-30e Primers

MP-r00067 150 ul / 10 uM
EUR 176

rno-miR-30d Primers

MP-r00069 150 ul / 10 uM
EUR 176

rno-miR-30a Primers

MP-r00070 150 ul / 10 uM
EUR 176

rno-miR-31 Primers

MP-r00071 150 ul / 10 uM
EUR 176

rno-miR-32 Primers

MP-r00072 150 ul / 10 uM
EUR 176

rno-miR-33 Primers

MP-r00073 150 ul / 10 uM
EUR 176

rno-miR-34b Primers

MP-r00074 150 ul / 10 uM
EUR 176

rno-miR-34c Primers

MP-r00075 150 ul / 10 uM
EUR 176

rno-miR-34a Primers

MP-r00076 150 ul / 10 uM
EUR 176

rno-miR-92a Primers

MP-r00077 150 ul / 10 uM
EUR 176

rno-miR-93 Primers

MP-r00078 150 ul / 10 uM
EUR 176

rno-miR-96 Primers

MP-r00079 150 ul / 10 uM
EUR 176

rno-miR-98 Primers

MP-r00080 150 ul / 10 uM
EUR 176

rno-miR-99a Primers

MP-r00081 150 ul / 10 uM
EUR 176

rno-miR-99b Primers

MP-r00082 150 ul / 10 uM
EUR 176

rno-miR-100 Primers

MP-r00083 150 ul / 10 uM
EUR 176

rno-miR-101a Primers

MP-r00084 150 ul / 10 uM
EUR 176

rno-miR-103 Primers

MP-r00085 150 ul / 10 uM
EUR 176

rno-miR-106b Primers

MP-r00086 150 ul / 10 uM
EUR 176

rno-miR-107 Primers

MP-r00087 150 ul / 10 uM
EUR 176

rno-miR-122 Primers

MP-r00088 150 ul / 10 uM
EUR 176

rno-miR-124 Primers

MP-r00089 150 ul / 10 uM
EUR 176

rno-miR-126 Primers

MP-r00092 150 ul / 10 uM
EUR 176

rno-miR-127 Primers

MP-r00093 150 ul / 10 uM
EUR 176

rno-miR-128 Primers

MP-r00094 150 ul / 10 uM
EUR 176

rno-miR-130a Primers

MP-r00095 150 ul / 10 uM
EUR 176

rno-miR-130b Primers

MP-r00096 150 ul / 10 uM
EUR 176

rno-miR-132 Primers

MP-r00097 150 ul / 10 uM
EUR 176

rno-miR-133a Primers

MP-r00098 150 ul / 10 uM
EUR 176

rno-miR-134 Primers

MP-r00099 150 ul / 10 uM
EUR 176

rno-miR-135a Primers

MP-r00100 150 ul / 10 uM
EUR 176

rno-miR-136 Primers

MP-r00101 150 ul / 10 uM
EUR 176

rno-miR-137 Primers

MP-r00102 150 ul / 10 uM
EUR 176

rno-miR-138 Primers

MP-r00103 150 ul / 10 uM
EUR 176

rno-miR-141 Primers

MP-r00105 150 ul / 10 uM
EUR 176

rno-miR-143 Primers

MP-r00107 150 ul / 10 uM
EUR 176

rno-miR-144 Primers

MP-r00108 150 ul / 10 uM
EUR 176

rno-miR-145 Primers

MP-r00109 150 ul / 10 uM
EUR 176

rno-miR-146a Primers

MP-r00110 150 ul / 10 uM
EUR 176

rno-miR-150 Primers

MP-r00111 150 ul / 10 uM
EUR 176

rno-miR-152 Primers

MP-r00112 150 ul / 10 uM
EUR 176

rno-miR-153 Primers

MP-r00113 150 ul / 10 uM
EUR 176

rno-miR-154 Primers

MP-r00114 150 ul / 10 uM
EUR 176

rno-miR-181c Primers

MP-r00115 150 ul / 10 uM
EUR 176

rno-miR-181a Primers

MP-r00116 150 ul / 10 uM
EUR 176

rno-miR-181a Primers

MP-r00117 150 ul / 10 uM
EUR 176

rno-miR-181b Primers

MP-r00118 150 ul / 10 uM
EUR 176

rno-miR-183 Primers

MP-r00119 150 ul / 10 uM
EUR 176

rno-miR-184 Primers

MP-r00120 150 ul / 10 uM
EUR 176

rno-miR-185 Primers

MP-r00121 150 ul / 10 uM
EUR 176

rno-miR-186 Primers

MP-r00122 150 ul / 10 uM
EUR 176

rno-miR-187 Primers

MP-r00123 150 ul / 10 uM
EUR 176

rno-miR-190 Primers

MP-r00124 150 ul / 10 uM
EUR 176

rno-miR-191 Primers

MP-r00125 150 ul / 10 uM
EUR 176

rno-miR-192 Primers

MP-r00126 150 ul / 10 uM
EUR 176

rno-miR-193 Primers

MP-r00127 150 ul / 10 uM
EUR 176

rno-miR-194 Primers

MP-r00128 150 ul / 10 uM
EUR 176

rno-miR-195 Primers

MP-r00129 150 ul / 10 uM
EUR 176

rno-miR-196a Primers

MP-r00130 150 ul / 10 uM
EUR 176

rno-miR-200c Primers

MP-r00132 150 ul / 10 uM
EUR 176

rno-miR-200a Primers

MP-r00133 150 ul / 10 uM
EUR 176

rno-miR-200b Primers

MP-r00134 150 ul / 10 uM
EUR 176

rno-miR-203 Primers

MP-r00135 150 ul / 10 uM
EUR 176

rno-miR-204 Primers

MP-r00136 150 ul / 10 uM
EUR 176

rno-miR-205 Primers

MP-r00137 150 ul / 10 uM
EUR 176

rno-miR-206 Primers

MP-r00138 150 ul / 10 uM
EUR 176

rno-miR-208 Primers

MP-r00139 150 ul / 10 uM
EUR 176

rno-miR-210 Primers

MP-r00140 150 ul / 10 uM
EUR 176

rno-miR-211 Primers

MP-r00141 150 ul / 10 uM
EUR 176

rno-miR-212 Primers

MP-r00142 150 ul / 10 uM
EUR 176

rno-miR-214 Primers

MP-r00143 150 ul / 10 uM
EUR 176

rno-miR-216a Primers

MP-r00144 150 ul / 10 uM
EUR 176
Conclusion: Our NGS workflow based mostly on the Ion Torrent sequencer was profitable within the detection of huge deletions and non-deletional defects in thalassemia with excessive accuracy and repeatability.

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