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 unit 33x45 cm
ESEQ1100-SYS ea
EUR 1535
sequencing system 20x50 cm
ESEQ1200-SYS ea
EUR 1535
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
  • Product category: PCR Related/PCR Kits (Cleanup)
PCR Clean Up for DNA Sequencing
BT5101 1000Preps, 1000prep
EUR 461.08
  • Product category: PCR Related/PCR Kits (Cleanup)
DNA Library Prep Kit for IIlumina Sequencing
K1475-12 12 Rxns
EUR 480
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
Random Primers
S300 30 µg
EUR 46
Random Primers
S305 5x30 µg
EUR 102
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
mmu-miR-30f Primers
MPM01166 150 ul / 10 uM
EUR 121
mmu-miR-3154 Primers
MPM01188 150 ul / 10 uM
EUR 121
mmu-miR-344i Primers
MPM01226 150 ul / 10 uM
EUR 121
mmu-miR-3473b Primers
MPM01229 150 ul / 10 uM
EUR 121
mmu-miR-3473c Primers
MPM01230 150 ul / 10 uM
EUR 121
mmu-miR-3473d Primers
MPM01231 150 ul / 10 uM
EUR 121
mmu-miR-3473e Primers
MPM01232 150 ul / 10 uM
EUR 121
mmu-miR-3473f Primers
MPM01233 150 ul / 10 uM
EUR 121
mmu-miR-3473g Primers
MPM01234 150 ul / 10 uM
EUR 121
mmu-miR-3535 Primers
MPM01245 150 ul / 10 uM
EUR 121
mmu-miR-3552 Primers
MPM01250 150 ul / 10 uM
EUR 121
mmu-miR-378c Primers
MPM01282 150 ul / 10 uM
EUR 121
mmu-miR-378d Primers
MPM01283 150 ul / 10 uM
EUR 121
mmu-miR-451a Primers
MPM01313 150 ul / 10 uM
EUR 121
mmu-miR-451b Primers
MPM01314 150 ul / 10 uM
EUR 121
mmu-miR-466q Primers
MPM01321 150 ul / 10 uM
EUR 121
mmu-miR-496b Primers
MPM01360 150 ul / 10 uM
EUR 121
mmu-miR-497b Primers
MPM01363 150 ul / 10 uM
EUR 121
mmu-miR-5046 Primers
MPM01372 150 ul / 10 uM
EUR 121
mmu-miR-5098 Primers
MPM01373 150 ul / 10 uM
EUR 121
mmu-miR-5099 Primers
MPM01374 150 ul / 10 uM
EUR 121
mmu-miR-5100 Primers
MPM01375 150 ul / 10 uM
EUR 121
mmu-miR-5101 Primers
MPM01376 150 ul / 10 uM
EUR 121
mmu-miR-5103 Primers
MPM01377 150 ul / 10 uM
EUR 121
mmu-miR-5104 Primers
MPM01378 150 ul / 10 uM
EUR 121
mmu-miR-5106 Primers
MPM01379 150 ul / 10 uM
EUR 121
mmu-miR-5108 Primers
MPM01382 150 ul / 10 uM
EUR 121
mmu-miR-5110 Primers
MPM01383 150 ul / 10 uM
EUR 121
mmu-miR-5112 Primers
MPM01384 150 ul / 10 uM
EUR 121
mmu-miR-5113 Primers
MPM01385 150 ul / 10 uM
EUR 121
mmu-miR-5114 Primers
MPM01386 150 ul / 10 uM
EUR 121
mmu-miR-5116 Primers
MPM01387 150 ul / 10 uM
EUR 121
mmu-miR-5118 Primers
MPM01388 150 ul / 10 uM
EUR 121
mmu-miR-5119 Primers
MPM01389 150 ul / 10 uM
EUR 121
mmu-miR-5120 Primers
MPM01390 150 ul / 10 uM
EUR 121
mmu-miR-5121 Primers
MPM01391 150 ul / 10 uM
EUR 121
mmu-miR-5122 Primers
MPM01392 150 ul / 10 uM
EUR 121
mmu-miR-5123 Primers
MPM01393 150 ul / 10 uM
EUR 121
mmu-miR-5124a Primers
MPM01394 150 ul / 10 uM
EUR 121
mmu-miR-5124b Primers
MPM01395 150 ul / 10 uM
EUR 121
mmu-miR-5125 Primers
MPM01396 150 ul / 10 uM
EUR 121
mmu-miR-5126 Primers
MPM01397 150 ul / 10 uM
EUR 121
mmu-miR-5127 Primers
MPM01398 150 ul / 10 uM
EUR 121
mmu-miR-5128 Primers
MPM01399 150 ul / 10 uM
EUR 121
mmu-miR-5130 Primers
MPM01402 150 ul / 10 uM
EUR 121
mmu-miR-5131 Primers
MPM01403 150 ul / 10 uM
EUR 121
mmu-miR-5133 Primers
MPM01406 150 ul / 10 uM
EUR 121
mmu-miR-5134 Primers
MPM01407 150 ul / 10 uM
EUR 121
mmu-miR-5135 Primers
MPM01410 150 ul / 10 uM
EUR 121
mmu-miR-5136 Primers
MPM01411 150 ul / 10 uM
EUR 121
mmu-miR-5710 Primers
MPM01447 150 ul / 10 uM
EUR 121
mmu-miR-6236 Primers
MPM01454 150 ul / 10 uM
EUR 121
mmu-miR-6237 Primers
MPM01455 150 ul / 10 uM
EUR 121
mmu-miR-6238 Primers
MPM01456 150 ul / 10 uM
EUR 121
mmu-miR-6239 Primers
MPM01457 150 ul / 10 uM
EUR 121
mmu-miR-6240 Primers
MPM01458 150 ul / 10 uM
EUR 121
mmu-miR-6241 Primers
MPM01459 150 ul / 10 uM
EUR 121
mmu-miR-6243 Primers
MPM01460 150 ul / 10 uM
EUR 121
mmu-miR-6244 Primers
MPM01461 150 ul / 10 uM
EUR 121
mmu-miR-6335 Primers
MPM01462 150 ul / 10 uM
EUR 121
mmu-miR-6336 Primers
MPM01463 150 ul / 10 uM
EUR 121
mmu-miR-6337 Primers
MPM01464 150 ul / 10 uM
EUR 121
mmu-miR-6338 Primers
MPM01465 150 ul / 10 uM
EUR 121
mmu-miR-6339 Primers
MPM01466 150 ul / 10 uM
EUR 121
mmu-miR-6340 Primers
MPM01467 150 ul / 10 uM
EUR 121
mmu-miR-6341 Primers
MPM01468 150 ul / 10 uM
EUR 121
mmu-miR-6342 Primers
MPM01469 150 ul / 10 uM
EUR 121
mmu-miR-6343 Primers
MPM01470 150 ul / 10 uM
EUR 121
mmu-miR-6344 Primers
MPM01471 150 ul / 10 uM
EUR 121
mmu-miR-6345 Primers
MPM01472 150 ul / 10 uM
EUR 121
mmu-miR-6346 Primers
MPM01473 150 ul / 10 uM
EUR 121
mmu-miR-6347 Primers
MPM01474 150 ul / 10 uM
EUR 121
mmu-miR-6348 Primers
MPM01475 150 ul / 10 uM
EUR 121
mmu-miR-6349 Primers
MPM01476 150 ul / 10 uM
EUR 121
mmu-miR-6350 Primers
MPM01477 150 ul / 10 uM
EUR 121
mmu-miR-6351 Primers
MPM01478 150 ul / 10 uM
EUR 121
mmu-miR-6352 Primers
MPM01479 150 ul / 10 uM
EUR 121
mmu-miR-6353 Primers
MPM01480 150 ul / 10 uM
EUR 121
mmu-miR-6354 Primers
MPM01481 150 ul / 10 uM
EUR 121
mmu-miR-6355 Primers
MPM01482 150 ul / 10 uM
EUR 121
mmu-miR-6356 Primers
MPM01483 150 ul / 10 uM
EUR 121
mmu-miR-6357 Primers
MPM01484 150 ul / 10 uM
EUR 121
mmu-miR-6358 Primers
MPM01485 150 ul / 10 uM
EUR 121
mmu-miR-6359 Primers
MPM01486 150 ul / 10 uM
EUR 121
mmu-miR-6360 Primers
MPM01487 150 ul / 10 uM
EUR 121
mmu-miR-6361 Primers
MPM01488 150 ul / 10 uM
EUR 121
mmu-miR-6362 Primers
MPM01489 150 ul / 10 uM
EUR 121
mmu-miR-6363 Primers
MPM01490 150 ul / 10 uM
EUR 121
mmu-miR-6364 Primers
MPM01491 150 ul / 10 uM
EUR 121
mmu-miR-6365 Primers
MPM01492 150 ul / 10 uM
EUR 121
mmu-miR-6366 Primers
MPM01493 150 ul / 10 uM
EUR 121
mmu-miR-6367 Primers
MPM01494 150 ul / 10 uM
EUR 121
mmu-miR-6368 Primers
MPM01495 150 ul / 10 uM
EUR 121
mmu-miR-6369 Primers
MPM01496 150 ul / 10 uM
EUR 121
mmu-miR-6370 Primers
MPM01497 150 ul / 10 uM
EUR 121
mmu-miR-6371 Primers
MPM01498 150 ul / 10 uM
EUR 121
mmu-miR-6372 Primers
MPM01499 150 ul / 10 uM
EUR 121
mmu-miR-6373 Primers
MPM01500 150 ul / 10 uM
EUR 121
mmu-miR-6374 Primers
MPM01501 150 ul / 10 uM
EUR 121
mmu-miR-6375 Primers
MPM01502 150 ul / 10 uM
EUR 121
mmu-miR-6376 Primers
MPM01503 150 ul / 10 uM
EUR 121
mmu-miR-6377 Primers
MPM01504 150 ul / 10 uM
EUR 121
mmu-miR-6378 Primers
MPM01505 150 ul / 10 uM
EUR 121
mmu-miR-6379 Primers
MPM01506 150 ul / 10 uM
EUR 121
mmu-miR-6380 Primers
MPM01507 150 ul / 10 uM
EUR 121
mmu-miR-6381 Primers
MPM01508 150 ul / 10 uM
EUR 121
mmu-miR-6382 Primers
MPM01509 150 ul / 10 uM
EUR 121
mmu-miR-6383 Primers
MPM01510 150 ul / 10 uM
EUR 121
mmu-miR-6384 Primers
MPM01511 150 ul / 10 uM
EUR 121
mmu-miR-6385 Primers
MPM01512 150 ul / 10 uM
EUR 121
mmu-miR-6386 Primers
MPM01513 150 ul / 10 uM
EUR 121
mmu-miR-6387 Primers
MPM01514 150 ul / 10 uM
EUR 121
mmu-miR-6388 Primers
MPM01515 150 ul / 10 uM
EUR 121
mmu-miR-6389 Primers
MPM01516 150 ul / 10 uM
EUR 121
mmu-miR-6390 Primers
MPM01517 150 ul / 10 uM
EUR 121
mmu-miR-6391 Primers
MPM01518 150 ul / 10 uM
EUR 121
mmu-miR-6393 Primers
MPM01521 150 ul / 10 uM
EUR 121
mmu-miR-6394 Primers
MPM01522 150 ul / 10 uM
EUR 121
mmu-miR-6395 Primers
MPM01523 150 ul / 10 uM
EUR 121
mmu-miR-6396 Primers
MPM01524 150 ul / 10 uM
EUR 121
mmu-miR-6397 Primers
MPM01525 150 ul / 10 uM
EUR 121
mmu-miR-6398 Primers
MPM01526 150 ul / 10 uM
EUR 121
mmu-miR-6399 Primers
MPM01527 150 ul / 10 uM
EUR 121
mmu-miR-6400 Primers
MPM01528 150 ul / 10 uM
EUR 121
mmu-miR-6401 Primers
MPM01529 150 ul / 10 uM
EUR 121
mmu-miR-6402 Primers
MPM01530 150 ul / 10 uM
EUR 121
mmu-miR-6403 Primers
MPM01531 150 ul / 10 uM
EUR 121
mmu-miR-6404 Primers
MPM01532 150 ul / 10 uM
EUR 121
mmu-miR-6405 Primers
MPM01533 150 ul / 10 uM
EUR 121
mmu-miR-6406 Primers
MPM01534 150 ul / 10 uM
EUR 121
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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