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.

sequencing system 20x50 cm

ESEQ1200-SYS ea
EUR 1842

Trypin for Mass & Sequencing

T9600-025 25ug
EUR 174

Trypin for Mass & Sequencing

T9600-100 100ug
EUR 262.8

Trypin for Mass & Sequencing

T9600-112 12x100ug
EUR 1988.4

Trypin for Mass & Sequencing

T9600-400 4x100ug
EUR 781.2

EpiNext Bisulfite Sequencing Kit (Illumina)

P-1056-12 12 Reactions
EUR 625.9

EpiNext Bisulfite Sequencing Kit (Illumina)

P-1056-24 24 Reactions
EUR 1225.4

SequaGel Sequencing System 1L Kit

NAT1136 1KIT
EUR 127.68

SequaGel Sequencing System 2.2L Kit

NAT1138 EACH
EUR 217.74

EpiNext Bisulfite Sequencing Kit (Illumina)

P-1056
  • EUR 1798.26
  • EUR 625.90
  • EUR 1225.40
  • 24 Reactions
  • 12 Reactions
  • 24 Reactions

PCR Clean Up for DNA Sequencing

BT5100 100preps
EUR 114.82

PCR Clean Up for DNA Sequencing

BT5101 1000Preps, 1000prep
EUR 553.3

DNA Library Prep Kit for IIlumina Sequencing

K1475-12 12 Rxns
EUR 576

MagaDye™ 4 Color Sanger Sequencing Terminator Kit

17068 5 nmoles
EUR 2091

RNU43 Primers

MPM00003 150 ul / 10 uM
EUR 145.2

snoRNA142 Primers

MPM00004 150 ul / 10 uM
EUR 145.2

U1 Primers

MP00001 150 ul / 10 uM
EUR 145.2

SNORD44 Primers

MPH00003 150 ul / 10 uM
EUR 145.2

SNORD47 Primers

MPH00004 150 ul / 10 uM
EUR 145.2

SNORD48 Primers

MPH00005 150 ul / 10 uM
EUR 145.2

Random Primers

S300 30 µg
EUR 55.2

Random Primers

S305 5x30 µg
EUR 122.4

U6 snRNA Primers

MPM00002 150 ul / 10 uM
EUR 145.2

U1 snRNA Primers

MPM00006 150 ul / 10 uM
EUR 145.2

U6-2 Primers

MPH00001 150 ul / 10 uM
EUR 145.2

rno-miR-323 Primers

MP-r00001 150 ul / 10 uM
EUR 211.2

rno-miR-301a Primers

MP-r00002 150 ul / 10 uM
EUR 211.2

rno-miR-326 Primers

MP-r00005 150 ul / 10 uM
EUR 211.2

rno-miR-327 Primers

MP-r00006 150 ul / 10 uM
EUR 211.2

rno-let-7d Primers

MP-r00007 150 ul / 10 uM
EUR 211.2

rno-miR-328 Primers

MP-r00008 150 ul / 10 uM
EUR 211.2

rno-miR-329 Primers

MP-r00009 150 ul / 10 uM
EUR 211.2

rno-miR-331 Primers

MP-r00010 150 ul / 10 uM
EUR 211.2

rno-miR-140 Primers

MP-r00011 150 ul / 10 uM
EUR 211.2

rno-miR-335 Primers

MP-r00012 150 ul / 10 uM
EUR 211.2

rno-miR-336 Primers

MP-r00013 150 ul / 10 uM
EUR 211.2

rno-miR-337 Primers

MP-r00014 150 ul / 10 uM
EUR 211.2

rno-miR-338 Primers

MP-r00015 150 ul / 10 uM
EUR 211.2

rno-miR-341 Primers

MP-r00017 150 ul / 10 uM
EUR 211.2

rno-miR-343 Primers

MP-r00018 150 ul / 10 uM
EUR 211.2

rno-miR-346 Primers

MP-r00020 150 ul / 10 uM
EUR 211.2

rno-miR-347 Primers

MP-r00021 150 ul / 10 uM
EUR 211.2

rno-miR-349 Primers

MP-r00022 150 ul / 10 uM
EUR 211.2

rno-miR-129 Primers

MP-r00023 150 ul / 10 uM
EUR 211.2

rno-miR-20a Primers

MP-r00024 150 ul / 10 uM
EUR 211.2

rno-miR-350 Primers

MP-r00025 150 ul / 10 uM
EUR 211.2

rno-miR-7a Primers

MP-r00026 150 ul / 10 uM
EUR 211.2

rno-miR-7a Primers

MP-r00027 150 ul / 10 uM
EUR 211.2

rno-miR-351 Primers

MP-r00028 150 ul / 10 uM
EUR 211.2

rno-miR-352 Primers

MP-r00029 150 ul / 10 uM
EUR 211.2

rno-miR-135b Primers

MP-r00030 150 ul / 10 uM
EUR 211.2

rno-miR-151 Primers

MP-r00031 150 ul / 10 uM
EUR 211.2

rno-miR-101b Primers

MP-r00032 150 ul / 10 uM
EUR 211.2

rno-let-7a Primers

MP-r00033 150 ul / 10 uM
EUR 211.2

rno-let-7b Primers

MP-r00034 150 ul / 10 uM
EUR 211.2

rno-let-7c Primers

MP-r00035 150 ul / 10 uM
EUR 211.2

rno-let-7e Primers

MP-r00036 150 ul / 10 uM
EUR 211.2

rno-let-7f Primers

MP-r00037 150 ul / 10 uM
EUR 211.2

rno-let-7i Primers

MP-r00038 150 ul / 10 uM
EUR 211.2

rno-miR-7b Primers

MP-r00039 150 ul / 10 uM
EUR 211.2

rno-miR-9 Primers

MP-r00040 150 ul / 10 uM
EUR 211.2

rno-miR-10b Primers

MP-r00042 150 ul / 10 uM
EUR 211.2

rno-miR-15b Primers

MP-r00043 150 ul / 10 uM
EUR 211.2

rno-miR-16 Primers

MP-r00044 150 ul / 10 uM
EUR 211.2

rno-miR-18a Primers

MP-r00047 150 ul / 10 uM
EUR 211.2

rno-miR-19b Primers

MP-r00048 150 ul / 10 uM
EUR 211.2

rno-miR-19a Primers

MP-r00049 150 ul / 10 uM
EUR 211.2

rno-miR-21 Primers

MP-r00050 150 ul / 10 uM
EUR 211.2

rno-miR-22 Primers

MP-r00051 150 ul / 10 uM
EUR 211.2

rno-miR-23a Primers

MP-r00052 150 ul / 10 uM
EUR 211.2

rno-miR-23b Primers

MP-r00053 150 ul / 10 uM
EUR 211.2

rno-miR-24 Primers

MP-r00054 150 ul / 10 uM
EUR 211.2

rno-miR-24 Primers

MP-r00055 150 ul / 10 uM
EUR 211.2

rno-miR-25 Primers

MP-r00056 150 ul / 10 uM
EUR 211.2

rno-miR-26a Primers

MP-r00057 150 ul / 10 uM
EUR 211.2

rno-miR-26b Primers

MP-r00058 150 ul / 10 uM
EUR 211.2

rno-miR-27b Primers

MP-r00059 150 ul / 10 uM
EUR 211.2

rno-miR-27a Primers

MP-r00060 150 ul / 10 uM
EUR 211.2

rno-miR-28 Primers

MP-r00061 150 ul / 10 uM
EUR 211.2

rno-miR-29b Primers

MP-r00062 150 ul / 10 uM
EUR 211.2

rno-miR-29b Primers

MP-r00063 150 ul / 10 uM
EUR 211.2

rno-miR-29a Primers

MP-r00064 150 ul / 10 uM
EUR 211.2

rno-miR-29c Primers

MP-r00065 150 ul / 10 uM
EUR 211.2

rno-miR-30c Primers

MP-r00066 150 ul / 10 uM
EUR 211.2

rno-miR-30e Primers

MP-r00067 150 ul / 10 uM
EUR 211.2

rno-miR-30d Primers

MP-r00069 150 ul / 10 uM
EUR 211.2

rno-miR-30a Primers

MP-r00070 150 ul / 10 uM
EUR 211.2

rno-miR-31 Primers

MP-r00071 150 ul / 10 uM
EUR 211.2

rno-miR-32 Primers

MP-r00072 150 ul / 10 uM
EUR 211.2

rno-miR-33 Primers

MP-r00073 150 ul / 10 uM
EUR 211.2

rno-miR-34b Primers

MP-r00074 150 ul / 10 uM
EUR 211.2

rno-miR-34c Primers

MP-r00075 150 ul / 10 uM
EUR 211.2

rno-miR-34a Primers

MP-r00076 150 ul / 10 uM
EUR 211.2

rno-miR-92a Primers

MP-r00077 150 ul / 10 uM
EUR 211.2

rno-miR-93 Primers

MP-r00078 150 ul / 10 uM
EUR 211.2

rno-miR-96 Primers

MP-r00079 150 ul / 10 uM
EUR 211.2

rno-miR-98 Primers

MP-r00080 150 ul / 10 uM
EUR 211.2

rno-miR-99a Primers

MP-r00081 150 ul / 10 uM
EUR 211.2

rno-miR-99b Primers

MP-r00082 150 ul / 10 uM
EUR 211.2

rno-miR-100 Primers

MP-r00083 150 ul / 10 uM
EUR 211.2

rno-miR-101a Primers

MP-r00084 150 ul / 10 uM
EUR 211.2

hsa-miR-4672 Primers

MPH02854 150 ul / 10 uM
EUR 145.2

hsa-miR-4673 Primers

MPH02855 150 ul / 10 uM
EUR 145.2

hsa-miR-4674 Primers

MPH02856 150 ul / 10 uM
EUR 145.2

hsa-miR-4675 Primers

MPH02857 150 ul / 10 uM
EUR 145.2

hsa-miR-4678 Primers

MPH02862 150 ul / 10 uM
EUR 145.2

hsa-miR-4679 Primers

MPH02863 150 ul / 10 uM
EUR 145.2

hsa-miR-4681 Primers

MPH02866 150 ul / 10 uM
EUR 145.2

hsa-miR-4682 Primers

MPH02867 150 ul / 10 uM
EUR 145.2

hsa-miR-4683 Primers

MPH02868 150 ul / 10 uM
EUR 145.2

hsa-miR-4686 Primers

MPH02873 150 ul / 10 uM
EUR 145.2

hsa-miR-4688 Primers

MPH02876 150 ul / 10 uM
EUR 145.2

hsa-miR-4689 Primers

MPH02877 150 ul / 10 uM
EUR 145.2

hsa-miR-4692 Primers

MPH02882 150 ul / 10 uM
EUR 145.2

hsa-miR-4696 Primers

MPH02889 150 ul / 10 uM
EUR 145.2

hsa-miR-4698 Primers

MPH02892 150 ul / 10 uM
EUR 145.2

hsa-miR-4705 Primers

MPH02903 150 ul / 10 uM
EUR 145.2

hsa-miR-4706 Primers

MPH02904 150 ul / 10 uM
EUR 145.2

hsa-miR-4710 Primers

MPH02911 150 ul / 10 uM
EUR 145.2

hsa-miR-4718 Primers

MPH02926 150 ul / 10 uM
EUR 145.2

hsa-miR-4719 Primers

MPH02927 150 ul / 10 uM
EUR 145.2

hsa-miR-4721 Primers

MPH02930 150 ul / 10 uM
EUR 145.2

hsa-miR-4729 Primers

MPH02945 150 ul / 10 uM
EUR 145.2

hsa-miR-4730 Primers

MPH02946 150 ul / 10 uM
EUR 145.2

hsa-miR-4734 Primers

MPH02953 150 ul / 10 uM
EUR 145.2

hsa-miR-4736 Primers

MPH02956 150 ul / 10 uM
EUR 145.2

hsa-miR-4737 Primers

MPH02957 150 ul / 10 uM
EUR 145.2

hsa-miR-4739 Primers

MPH02960 150 ul / 10 uM
EUR 145.2

hsa-miR-4741 Primers

MPH02963 150 ul / 10 uM
EUR 145.2

hsa-miR-4744 Primers

MPH02968 150 ul / 10 uM
EUR 145.2

hsa-miR-4748 Primers

MPH02975 150 ul / 10 uM
EUR 145.2

hsa-miR-4751 Primers

MPH02980 150 ul / 10 uM
EUR 145.2

hsa-miR-4752 Primers

MPH02981 150 ul / 10 uM
EUR 145.2

hsa-miR-4754 Primers

MPH02984 150 ul / 10 uM
EUR 145.2

hsa-miR-4759 Primers

MPH02993 150 ul / 10 uM
EUR 145.2

hsa-miR-4765 Primers

MPH03004 150 ul / 10 uM
EUR 145.2

hsa-miR-4767 Primers

MPH03007 150 ul / 10 uM
EUR 145.2

hsa-miR-4770 Primers

MPH03012 150 ul / 10 uM
EUR 145.2

hsa-miR-4771 Primers

MPH03013 150 ul / 10 uM
EUR 145.2

hsa-miR-4773 Primers

MPH03016 150 ul / 10 uM
EUR 145.2

hsa-miR-4775 Primers

MPH03019 150 ul / 10 uM
EUR 145.2

hsa-miR-4779 Primers

MPH03026 150 ul / 10 uM
EUR 145.2

hsa-miR-4780 Primers

MPH03027 150 ul / 10 uM
EUR 145.2

hsa-miR-4784 Primers

MPH03034 150 ul / 10 uM
EUR 145.2

hsa-miR-4785 Primers

MPH03035 150 ul / 10 uM
EUR 145.2

hsa-miR-4788 Primers

MPH03040 150 ul / 10 uM
EUR 145.2

hsa-miR-4791 Primers

MPH03045 150 ul / 10 uM
EUR 145.2

hsa-miR-4792 Primers

MPH03046 150 ul / 10 uM
EUR 145.2

hsa-miR-4794 Primers

MPH03049 150 ul / 10 uM
EUR 145.2

hsa-miR-4801 Primers

MPH03062 150 ul / 10 uM
EUR 145.2

hsa-miR-4803 Primers

MPH03065 150 ul / 10 uM
EUR 145.2

hsa-miR-5047 Primers

MPH03119 150 ul / 10 uM
EUR 145.2
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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