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
RNU43 Primers
MPM00003 150 ul / 10 uM
EUR 121
snoRNA142 Primers
MPM00004 150 ul / 10 uM
EUR 121
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
Random Primers
S300 30 µg
EUR 46
Random Primers
S305 5x30 µg
EUR 102
U6 snRNA Primers
MPM00002 150 ul / 10 uM
EUR 121
U1 snRNA Primers
MPM00006 150 ul / 10 uM
EUR 121
U6-2 Primers
MPH00001 150 ul / 10 uM
EUR 121
mmu-miR-1931 Primers
MPM00151 150 ul / 10 uM
EUR 121
mmu-miR-1932 Primers
MPM00152 150 ul / 10 uM
EUR 121
mmu-miR-1934 Primers
MPM00155 150 ul / 10 uM
EUR 121
mmu-miR-1935 Primers
MPM00156 150 ul / 10 uM
EUR 121
mmu-miR-1936 Primers
MPM00157 150 ul / 10 uM
EUR 121
mmu-miR-1937a Primers
MPM00158 150 ul / 10 uM
EUR 121
mmu-miR-1937b Primers
MPM00159 150 ul / 10 uM
EUR 121
mmu-miR-1937c Primers
MPM00160 150 ul / 10 uM
EUR 121
mmu-miR-1938 Primers
MPM00161 150 ul / 10 uM
EUR 121
mmu-miR-1939 Primers
MPM00162 150 ul / 10 uM
EUR 121
mmu-miR-193b Primers
MPM00163 150 ul / 10 uM
EUR 121
mmu-miR-194 Primers
MPM00164 150 ul / 10 uM
EUR 121
mmu-miR-1940 Primers
MPM00165 150 ul / 10 uM
EUR 121
mmu-miR-1942 Primers
MPM00168 150 ul / 10 uM
EUR 121
mmu-miR-1943 Primers
MPM00169 150 ul / 10 uM
EUR 121
mmu-miR-1944 Primers
MPM00170 150 ul / 10 uM
EUR 121
mmu-miR-1945 Primers
MPM00171 150 ul / 10 uM
EUR 121
mmu-miR-1946a Primers
MPM00172 150 ul / 10 uM
EUR 121
mmu-miR-1946b Primers
MPM00173 150 ul / 10 uM
EUR 121
mmu-miR-1947 Primers
MPM00174 150 ul / 10 uM
EUR 121
mmu-miR-1948 Primers
MPM00175 150 ul / 10 uM
EUR 121
mmu-miR-1949 Primers
MPM00176 150 ul / 10 uM
EUR 121
mmu-miR-195 Primers
MPM00177 150 ul / 10 uM
EUR 121
mmu-miR-1950 Primers
MPM00178 150 ul / 10 uM
EUR 121
mmu-miR-1951 Primers
MPM00179 150 ul / 10 uM
EUR 121
mmu-miR-1952 Primers
MPM00180 150 ul / 10 uM
EUR 121
mmu-miR-1953 Primers
MPM00181 150 ul / 10 uM
EUR 121
mmu-miR-1954 Primers
MPM00182 150 ul / 10 uM
EUR 121
mmu-miR-1956 Primers
MPM00185 150 ul / 10 uM
EUR 121
mmu-miR-1957 Primers
MPM00186 150 ul / 10 uM
EUR 121
mmu-miR-1958 Primers
MPM00187 150 ul / 10 uM
EUR 121
mmu-miR-1959 Primers
MPM00188 150 ul / 10 uM
EUR 121
mmu-miR-1960 Primers
MPM00189 150 ul / 10 uM
EUR 121
mmu-miR-1961 Primers
MPM00190 150 ul / 10 uM
EUR 121
mmu-miR-1962 Primers
MPM00191 150 ul / 10 uM
EUR 121
mmu-miR-1963 Primers
MPM00192 150 ul / 10 uM
EUR 121
mmu-miR-1965 Primers
MPM00195 150 ul / 10 uM
EUR 121
mmu-miR-1966 Primers
MPM00196 150 ul / 10 uM
EUR 121
mmu-miR-1967 Primers
MPM00197 150 ul / 10 uM
EUR 121
mmu-miR-1968 Primers
MPM00198 150 ul / 10 uM
EUR 121
mmu-miR-1969 Primers
MPM00199 150 ul / 10 uM
EUR 121
mmu-miR-196a Primers
MPM00200 150 ul / 10 uM
EUR 121
mmu-miR-196b Primers
MPM00201 150 ul / 10 uM
EUR 121
mmu-miR-197 Primers
MPM00202 150 ul / 10 uM
EUR 121
mmu-miR-1970 Primers
MPM00203 150 ul / 10 uM
EUR 121
mmu-miR-1971 Primers
MPM00204 150 ul / 10 uM
EUR 121
mmu-miR-1981 Primers
MPM00205 150 ul / 10 uM
EUR 121
mmu-miR-1982.1 Primers
MPM00206 150 ul / 10 uM
EUR 121
mmu-miR-1982.2 Primers
MPM00207 150 ul / 10 uM
EUR 121
mmu-miR-1983 Primers
MPM00208 150 ul / 10 uM
EUR 121
mmu-miR-199b Primers
MPM00211 150 ul / 10 uM
EUR 121
mmu-miR-19a Primers
MPM00212 150 ul / 10 uM
EUR 121
mmu-miR-19b Primers
MPM00213 150 ul / 10 uM
EUR 121
mmu-miR-200a Primers
MPM00214 150 ul / 10 uM
EUR 121
mmu-miR-200b Primers
MPM00215 150 ul / 10 uM
EUR 121
mmu-miR-200c Primers
MPM00216 150 ul / 10 uM
EUR 121
mmu-miR-201 Primers
MPM00217 150 ul / 10 uM
EUR 121
mmu-miR-203 Primers
MPM00220 150 ul / 10 uM
EUR 121
mmu-miR-204 Primers
MPM00221 150 ul / 10 uM
EUR 121
mmu-miR-205 Primers
MPM00222 150 ul / 10 uM
EUR 121
mmu-miR-206 Primers
MPM00223 150 ul / 10 uM
EUR 121
mmu-miR-207 Primers
MPM00224 150 ul / 10 uM
EUR 121
mmu-miR-208b Primers
MPM00227 150 ul / 10 uM
EUR 121
mmu-miR-20a Primers
MPM00228 150 ul / 10 uM
EUR 121
mmu-miR-20b Primers
MPM00229 150 ul / 10 uM
EUR 121
mmu-miR-21 Primers
MPM00230 150 ul / 10 uM
EUR 121
mmu-miR-210 Primers
MPM00231 150 ul / 10 uM
EUR 121
mmu-miR-211 Primers
MPM00232 150 ul / 10 uM
EUR 121
mmu-miR-2136 Primers
MPM00235 150 ul / 10 uM
EUR 121
mmu-miR-2137 Primers
MPM00236 150 ul / 10 uM
EUR 121
mmu-miR-2139 Primers
MPM00237 150 ul / 10 uM
EUR 121
mmu-miR-214 Primers
MPM00238 150 ul / 10 uM
EUR 121
mmu-miR-2142 Primers
MPM00239 150 ul / 10 uM
EUR 121
mmu-miR-2143 Primers
MPM00240 150 ul / 10 uM
EUR 121
mmu-miR-2144 Primers
MPM00241 150 ul / 10 uM
EUR 121
mmu-miR-2145 Primers
MPM00242 150 ul / 10 uM
EUR 121
mmu-miR-215 Primers
MPM00243 150 ul / 10 uM
EUR 121
mmu-miR-216a Primers
MPM00244 150 ul / 10 uM
EUR 121
mmu-miR-216b Primers
MPM00245 150 ul / 10 uM
EUR 121
mmu-miR-217 Primers
MPM00246 150 ul / 10 uM
EUR 121
mmu-miR-218 Primers
MPM00247 150 ul / 10 uM
EUR 121
mmu-miR-2182 Primers
MPM00248 150 ul / 10 uM
EUR 121
mmu-miR-2183 Primers
MPM00249 150 ul / 10 uM
EUR 121
mmu-miR-22 Primers
MPM00252 150 ul / 10 uM
EUR 121
mmu-miR-221 Primers
MPM00253 150 ul / 10 uM
EUR 121
mmu-miR-222 Primers
MPM00254 150 ul / 10 uM
EUR 121
mmu-miR-223 Primers
MPM00255 150 ul / 10 uM
EUR 121
mmu-miR-224 Primers
MPM00256 150 ul / 10 uM
EUR 121
mmu-miR-23a Primers
MPM00257 150 ul / 10 uM
EUR 121
mmu-miR-23b Primers
MPM00258 150 ul / 10 uM
EUR 121
mmu-miR-24 Primers
MPM00259 150 ul / 10 uM
EUR 121
mmu-miR-25 Primers
MPM00260 150 ul / 10 uM
EUR 121
mmu-miR-26a Primers
MPM00261 150 ul / 10 uM
EUR 121
mmu-miR-26b Primers
MPM00262 150 ul / 10 uM
EUR 121
mmu-miR-27a Primers
MPM00263 150 ul / 10 uM
EUR 121
mmu-miR-27b Primers
MPM00264 150 ul / 10 uM
EUR 121
mmu-miR-28 Primers
MPM00265 150 ul / 10 uM
EUR 121
mmu-miR-2861 Primers
MPM00266 150 ul / 10 uM
EUR 121
mmu-miR-293 Primers
MPM00275 150 ul / 10 uM
EUR 121
mmu-miR-294 Primers
MPM00276 150 ul / 10 uM
EUR 121
mmu-miR-295 Primers
MPM00277 150 ul / 10 uM
EUR 121
mmu-miR-297a Primers
MPM00280 150 ul / 10 uM
EUR 121
mmu-miR-297c Primers
MPM00283 150 ul / 10 uM
EUR 121
mmu-miR-298 Primers
MPM00284 150 ul / 10 uM
EUR 121
mmu-miR-299 Primers
MPM00285 150 ul / 10 uM
EUR 121
mmu-miR-29a Primers
MPM00286 150 ul / 10 uM
EUR 121
mmu-miR-29b Primers
MPM00287 150 ul / 10 uM
EUR 121
mmu-miR-29c Primers
MPM00288 150 ul / 10 uM
EUR 121
mmu-miR-300 Primers
MPM00289 150 ul / 10 uM
EUR 121
mmu-miR-301a Primers
MPM00290 150 ul / 10 uM
EUR 121
mmu-miR-301b Primers
MPM00291 150 ul / 10 uM
EUR 121
mmu-miR-302a Primers
MPM00292 150 ul / 10 uM
EUR 121
mmu-miR-302b Primers
MPM00293 150 ul / 10 uM
EUR 121
mmu-miR-302c Primers
MPM00294 150 ul / 10 uM
EUR 121
mmu-miR-302d Primers
MPM00295 150 ul / 10 uM
EUR 121
mmu-miR-3058 Primers
MPM00298 150 ul / 10 uM
EUR 121
mmu-miR-3059 Primers
MPM00299 150 ul / 10 uM
EUR 121
mmu-miR-3060 Primers
MPM00300 150 ul / 10 uM
EUR 121
mmu-miR-3062 Primers
MPM00303 150 ul / 10 uM
EUR 121
mmu-miR-3063 Primers
MPM00304 150 ul / 10 uM
EUR 121
mmu-miR-3065 Primers
MPM00307 150 ul / 10 uM
EUR 121
mmu-miR-3066 Primers
MPM00308 150 ul / 10 uM
EUR 121
mmu-miR-3067 Primers
MPM00309 150 ul / 10 uM
EUR 121
mmu-miR-3068 Primers
MPM00310 150 ul / 10 uM
EUR 121
mmu-miR-3070a Primers
MPM00313 150 ul / 10 uM
EUR 121
mmu-miR-3071 Primers
MPM00316 150 ul / 10 uM
EUR 121
mmu-miR-3075 Primers
MPM00322 150 ul / 10 uM
EUR 121
mmu-miR-3077 Primers
MPM00325 150 ul / 10 uM
EUR 121
mmu-miR-3078 Primers
MPM00326 150 ul / 10 uM
EUR 121
mmu-miR-3081 Primers
MPM00331 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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