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.

Oligo, Sequencing Primer, SK (17mer)

MBS634188-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, SK (17mer)

MBS634188-5x0033mg 5x0.033mg
EUR 1235

Oligo, Sequencing Primer, KS (17mer)

MBS634192-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, KS (17mer)

MBS634192-5x0033mg 5x0.033mg
EUR 1235

Oligo, Sequencing Primer, SK (20mer)

MBS634219-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, SK (20mer)

MBS634219-5x0033mg 5x0.033mg
EUR 1240

Oligo, Sequencing Primer, T3 (17mer)

MBS634243-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, T3 (17mer)

MBS634243-5x0033mg 5x0.033mg
EUR 1235

Oligo, Sequencing Primer, T7 (17mer)

MBS634247-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, T7 (17mer)

MBS634247-5x0033mg 5x0.033mg
EUR 1235

Oligo, Sequencing Primer, KS (19mer)

MBS634259-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, KS (19mer)

MBS634259-5x0033mg 5x0.033mg
EUR 1235

Oligo, Sequencing Primer, T3 (20mer)

MBS634261-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, T3 (20mer)

MBS634261-5x0033mg 5x0.033mg
EUR 1240

Oligo, Sequencing Primer, T7 Universal (20mer)

MBS634194-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, T7 Universal (20mer)

MBS634194-5x0033mg 5x0.033mg
EUR 1240

Oligo, Sequencing Primer, SP6 Promoter (19mer)

MBS634260-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, SP6 Promoter (19mer)

MBS634260-5x0033mg 5x0.033mg
EUR 1235

Oligo, Sequencing Primer, Lambda gt11, Reverse (24mer)

MBS634120-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, Lambda gt11, Reverse (24mer)

MBS634120-5x0033mg 5x0.033mg
EUR 1245

Oligo, Sequencing Primer, Lambda gt10, Reverse (31mer)

MBS634167-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, Lambda gt10, Reverse (31mer)

MBS634167-5x0033mg 5x0.033mg
EUR 1245

Oligo, Sequencing Primer, Lambda gt11, Forward (24mer)

MBS634252-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, Lambda gt11, Forward (24mer)

MBS634252-5x0033mg 5x0.033mg
EUR 1245

Oligo, Sequencing Primer, Lambda gt10, Forward (27mer)

MBS634255-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, Lambda gt10, Forward (27mer)

MBS634255-5x0033mg 5x0.033mg
EUR 1240

Oligo, Sequencing Primer, pBR322 (EcoR I), Clockwise (16mer)

MBS634158-0033mg 0.033mg
EUR 305

Oligo, Sequencing Primer, pBR322 (EcoR I), Clockwise (16mer)

MBS634158-5x0033mg 5x0.033mg
EUR 1230

Oligo, Sequencing Primer, pBR322 (BamH I), Clockwise (20mer)

MBS634234-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, pBR322 (BamH I), Clockwise (20mer)

MBS634234-5x0033mg 5x0.033mg
EUR 1240

Oligo, Sequencing Primer, pBR322 (Pst I), Counterclockwise (16mer)

MBS634143-0033mg 0.033mg
EUR 305

Oligo, Sequencing Primer, pBR322 (Pst I), Counterclockwise (16mer)

MBS634143-5x0033mg 5x0.033mg
EUR 1230

Oligo, Sequencing Primer, pBR322 (BamH I), Counterclockwise (16mer)

MBS634262-0033mg 0.033mg
EUR 305

Oligo, Sequencing Primer, pBR322 (BamH I), Counterclockwise (16mer)

MBS634262-5x0033mg 5x0.033mg
EUR 1230

Oligo, Sequencing Primer, pBR322 (Hind III), Counterclockwise (16mer)

MBS634154-0033mg 0.033mg
EUR 305

Oligo, Sequencing Primer, pBR322 (Hind III), Counterclockwise (16mer)

MBS634154-5x0033mg 5x0.033mg
EUR 1230

Oligo, Sequencing Primer, M13 Reverse (16mer) [5'-AAC AGC TAT GAC CAT G-3']

MBS634131-0033mg 0.033mg
EUR 305

Oligo, Sequencing Primer, M13 Reverse (16mer) [5'-AAC AGC TAT GAC CAT G-3']

MBS634131-5x0033mg 5x0.033mg
EUR 1230

Oligo, Sequencing Primer, M13 (-20) Forward (17mer) [5'-GTA AAA CGA CGG CCA GT-3']

MBS634146-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, M13 (-20) Forward (17mer) [5'-GTA AAA CGA CGG CCA GT-3']

MBS634146-5x0033mg 5x0.033mg
EUR 1235

Oligo, Sequencing Primer, M13 Reverse (19mer) [5'-GGA AAC AGC TAT GAC CAT G-3']

MBS634148-0033mg 0.033mg
EUR 310

Oligo, Sequencing Primer, M13 Reverse (19mer) [5'-GGA AAC AGC TAT GAC CAT G-3']

MBS634148-5x0033mg 5x0.033mg
EUR 1235

sequencing unit 33x45 cm

ESEQ1100-SYS ea
EUR 1842

Sequencing Grade Chymotrypsin

RE013 100ug
EUR 309

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

sequencing system 20x50 cm

ESEQ1200-SYS ea
EUR 1842

Sequencing Grade V8 Protease

RE014 50ug
EUR 209

Chymotrypsin for Sequencing grade

C4001-010 4x25ug
EUR 375.6

Chymotrypsin for Sequencing grade

C4001-100 100ug
EUR 343.2

Sequencing Grade Modified Trypsin

RE012 100ug
EUR 229

Sequencing Grade Carboxypeptidase B

RE011 100ug
EUR 99

PCR Clean Up for DNA Sequencing

BT5100 100preps
EUR 114.82

PCR Clean Up for DNA Sequencing

BT5101 1000Preps, 1000prep
EUR 553.3

ACES Sequencing Buffer 10X, pH 7.7

21420000-1 1 L
EUR 138.62

ACES Sequencing Buffer 10X, pH 7.7

21420000-2 4 L
EUR 456.17

TBE-Buffer 5X for DNA Sequencing

40121072-1 100 mL
EUR 26.54

TBE-Buffer 5X for DNA Sequencing

40121072-2 500 mL
EUR 44.84

TBE-Buffer 5X for DNA Sequencing

40121072-3 1 L
EUR 72.29

TBE-Buffer 5X for DNA Sequencing

40121072-4 4 L
EUR 192.14

SequaGel Sequencing System 1L Kit - 1KIT

NAT1136 1KIT
EUR 187.65

SequaGel Sequencing System 2.2L Kit - EACH

NAT1138 EACH
EUR 318.6

Bovine Trypsin Purified Sequencing Grade

IBOTRYSEQ100UG each
EUR 148
Description: Bovine Trypsin Purified Sequencing Grade

Bovine Trypsin Purified Sequencing Grade

IBOTRYSEQ1MG each
EUR 765
Description: Bovine Trypsin Purified Sequencing Grade

Bovine Trypsin Purified Sequencing Grade

IBOTRYSEQ400UG each
EUR 419
Description: Bovine Trypsin Purified Sequencing Grade

Bovine Trypsin Purified Sequencing Grade

MBS136298-01mg 0.1mg
EUR 255

Bovine Trypsin Purified Sequencing Grade

MBS136298-04mg 0.4mg
EUR 540

Bovine Trypsin Purified Sequencing Grade

MBS136298-1mg 1mg
EUR 935

Bovine Trypsin Purified Sequencing Grade

MBS136298-5x1mg 5x1mg
EUR 4010

EpiNext Bisulfite Sequencing Kit (Illumina)

P-1056
  • Ask for price
  • Ask for price
  • Ask for price
  • 24 Reactions
  • 12 Reactions
  • 24 Reactions

DNA Library Prep Kit for IIlumina Sequencing

K1475-12 12 Rxns
EUR 576

Trifluoroacetic Acid (TFA) for HPLC & Sequencing, 99.9%

23947 10 x1 ml
EUR 165.03
Description: Part A

Bovine Trypsin Purified Sequencing Grade Lyophilized

IBOTRYSEQLY100UG each
EUR 130
Description: Bovine Trypsin Purified Sequencing Grade Lyophilized

Bovine Trypsin Purified Sequencing Grade Lyophilized

IBOTRYSEQLY1MG each
EUR 640
Description: Bovine Trypsin Purified Sequencing Grade Lyophilized

Bovine Trypsin Purified Sequencing Grade Lyophilized

IBOTRYSEQLY400UG each
EUR 344
Description: Bovine Trypsin Purified Sequencing Grade Lyophilized

Bovine Trypsin Purified Sequencing Grade Lyophilized

MBS136299-01mg 0.1mg
EUR 235

Bovine Trypsin Purified Sequencing Grade Lyophilized

MBS136299-04mg 0.4mg
EUR 465

Bovine Trypsin Purified Sequencing Grade Lyophilized

MBS136299-1mg 1mg
EUR 795

Bovine Trypsin Purified Sequencing Grade Lyophilized

MBS136299-5x1mg 5x1mg
EUR 3400

iLenti Sequence Primers

LV012 100 μl (10 μM)
EUR 75

MagaDye™ 4 Color Sanger Sequencing Terminator Kit

17068 5 nmoles
EUR 2091

Bovine Alpha Chymotrypsin 3X Crystallized TLCK Treated Sequencing Grade

IBOCHYTLCKSG100UG each
EUR 297
Description: Bovine Alpha Chymotrypsin 3X Crystallized TLCK Treated Sequencing Grade

Bovine Alpha Chymotrypsin 3X Crystallized TLCK Treated Sequencing Grade

MBS136223-01mg 0.1mg
EUR 400

Bovine Alpha Chymotrypsin 3X Crystallized TLCK Treated Sequencing Grade

MBS136223-5x01mg 5x0.1mg
EUR 1605

Bacteria-Derived Clostripain (Endoproteinase-Arg-C) Sequencing Grade Purified

IBCTCLOSGPF10UG each
EUR 165
Description: Bacteria-Derived Clostripain (Endoproteinase-Arg-C) Sequencing Grade Purified

Bacteria-Derived Clostripain (Endoproteinase-Arg-C) Sequencing Grade Purified

MBS136171-01mg 0.1mg
EUR 270

Bacteria-Derived Clostripain (Endoproteinase-Arg-C) Sequencing Grade Purified

MBS136171-5x001mg 5x0.01mg
EUR 1035

Bacteria-Derived V8 Protease (Endoprotease Glu-C) Sequencing Grade Lyophilized

IBCTV8SEQLY250UG each
EUR 784
Description: Bacteria-Derived V8 Protease (Endoprotease Glu-C) Sequencing Grade Lyophilized

Bacteria-Derived V8 Protease (Endoprotease Glu-C) Sequencing Grade Lyophilized

IBCTV8SEQLY50UG each
EUR 304
Description: Bacteria-Derived V8 Protease (Endoprotease Glu-C) Sequencing Grade Lyophilized

SEPTA MAT, FOR 96 WELL PCR PLATES, SILICONE, GREY, NONSTERILE, FOR ABI MULTI-CAPILLARY SEQUENCING INSTRUMENTS, BULK

AM-96-SEPTA-3100 10/pk
EUR 639.6
Description: Sealing Products; Sealing mats - Axygen

Human Stem Cell Primer Library

HSCL-I 1 set
EUR 540

Rat NFKappaB Primer Library

RNFKB-I 1 set
EUR 774

Rat Cytokine Primer Library I

RCA-I 1 set
EUR 657.6

Rat Cytokine Primer Library II

RCA-II 1 set
EUR 657.6

Human DNA Repair Primer Library I

HDRL-I 1 set
EUR 657.6

Human DNA Repair Primer Library II

HDRL-II 1 set
EUR 657.6

Human Cell Cycle Primer Library

HCC-I 1 set
EUR 540

Mouse Cell Cycle Primer Library

MCC-I 1 set
EUR 540

Mouse DNA Repair Primer Library I

MDRL-I 1 set
EUR 774

Mouse DNA Repair Primer Library II

MDRL-II 1 set
EUR 774

Rat Autophagy Primer Library

RATPL-I 1 set
EUR 774

Human Melanoma Primer Library

HMEL-I 1 set
EUR 657.6

Human NFKappaB Primer Library

HNFKB-I 1 set
EUR 540

Human Oncogene Primer Library

HONC-I 1 set
EUR 657.6

Human Cytokine Primer Library I

HCA-I 1 set
EUR 540

Human Cytokine Primer Library II

HCA-II 1 set
EUR 540

Mouse Cytokine Primer Library I

MCA-I 1 set
EUR 540

Mouse Cytokine Primer Library II

MCA-II 1 set
EUR 540

Mouse Oncogene Primer Library

MONC-I 1 set
EUR 657.6

Mouse NFKappaB Primer Library

MNFKB-I 1 set
EUR 540

Human Liver Cancer Primer Library

HLCPL-I 1 set
EUR 657.6

Human Lung Cancer PCR Primer Library

HLUCPL-I 1 set
EUR 657.6

Human Apoptosis Primer Library

HPA-I 1 set
EUR 540

Human Autophagy Primer Library

HATPL-I 1 set
EUR 540

Human Brain Cancer Primer Library

HBRC-I 1 set
EUR 657.6

Human Colon Cancer Primer Library I

HCCP-I 1 set
EUR 657.6

Human Colon Cancer Primer Library II

HCCP-II 1 set
EUR 657.6

Mouse Apoptosis Primer Library

MPA-I 1 set
EUR 540

Mouse Autophagy Primer Library

MATPL-I 1 set
EUR 540

Rat Bdnf Signaling Primer Library

RBDNF-I 1 set
EUR 774

Rat Gdnf Signaling Primer Library

RGDNF-I 1 set
EUR 774

Human Homeobox Gene Primer Library

HHOX-I 1 set
EUR 774

Human Ras Signaling Primer Library

HRAS-I 1 set
EUR 657.6

Human Schwann Cell PCR Primer Library

HSCH-I 1 set
EUR 657.6

Human TNF Signaling Primer Library

HTNF-I 1 set
EUR 657.6

Human p53 Signaling Primer Library

HTPS-I 1 set
EUR 540

Human WNT Signaling Primer Library

HWNT-I 1 set
EUR 657.6

Human BMP Signaling Primer Library

HBMP-I 1 set
EUR 657.6

Human Cell Adhesion Primer Library

HCAD-I 1 set
EUR 774

Mouse p53 Signaling Primer Library

MTPS-I 1 set
EUR 540

Mouse WNT Signaling Primer Library

MWNT-I 1 set
EUR 657.6

Mouse Ras Signaling Primer Library

MRAS-I 1 set
EUR 657.6

Mouse BMP Signaling Primer Library

MBMP-I 1 set
EUR 657.6

Human EGFR Signaling Primer Library

HEGFR-I 1 set
EUR 657.6

Human ERBB Signaling Primer Library

HERBB-I 1 set
EUR 657.6

Human Gastric Cancer Primer Library

HGCPL-I 1 set
EUR 657.6

Human GDNF Signaling Primer Library

HGDNF-I 1 set
EUR 657.6

Human IL17 Signaling Primer Library

HIL17-I 1 set
EUR 657.6

Human Necroptosis Primer Library

HNCR-I 1 set
EUR 657.6

Human Ovarian Cancer Primer Library

HOVCL-I 1 set
EUR 657.6

Human PDGF Signaling Primer Library

HPDGF-I 1 set
EUR 657.6

Human SMAD Signaling Primer Library

HSMAD-I 1 set
EUR 657.6

Human Tumor Immunity Primer Library

HTIMU-I 1 set
EUR 657.6

Human VEGF Signaling Primer Library

HVEGF-I 1 set
EUR 657.6

Human BDNF Signaling Primer Library

HBDNF-I 1 set
EUR 657.6

Human Bladder Cancer Primer Library

HBLCP-I 1 set
EUR 657.6

Human cAMP Signaling Primer Library

HCAMP-I 1 set
EUR 657.6

Human Cancer Pathway Primer Library

HCAN-I 1 set
EUR 657.6

Human CREB Signaling Primer Library

HCREB-I 1 set
EUR 657.6

Mouse Tumor Immunity Primer Library

MTIMU-I 1 set
EUR 657.6
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.

Leave a Comment