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 subsequent–technology 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.
Analysis of Ion Torrent subsequent–technology sequencing for thalassemia analysis
Introduction: To guage a next-generation sequencing (NGS) workflow within the screening and analysis of thalassemia.
Strategies: On 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.
Outcomes: Of 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.
Trypin for Mass & Sequencing |
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| T9600-100 | GenDepot | 100ug | EUR 262.8 |
Trypin for Mass & Sequencing |
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Trypin for Mass & Sequencing |
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PCR Clean Up for DNA Sequencing |
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| BT5101 | Bio Basic | 1000Preps, 1000prep | EUR 553.3 |
EpiNext Bisulfite Sequencing Kit (Illumina) |
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DNA Library Prep Kit for IIlumina Sequencing |
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| K1475-12 | Biovision | 12 Rxns | EUR 576 |
MagaDye™ 4 Color Sanger Sequencing Terminator Kit |
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| 17068 | AAT Bioquest | 5 nmoles | EUR 2091 |
SEPTA MAT, FOR 96 WELL PCR PLATES, SILICONE, GREY, NONSTERILE, FOR ABI MULTI-CAPILLARY SEQUENCING INSTRUMENTS, BULK |
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Description: Sealing Products; Sealing mats - Axygen |
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Human Stem Cell Primer Library |
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Rat Cytokine Primer Library I |
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Mouse Cell Cycle Primer Library |
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Mouse Cytokine Primer Library I |
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Mouse Cytokine Primer Library II |
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| MCA-II | Real Time Primers | 1 set | EUR 540 |
Mouse NFKappaB Primer Library |
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| MNFKB-I | Real Time Primers | 1 set | EUR 540 |
Human Cytokine Primer Library I |
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| HCA-I | Real Time Primers | 1 set | EUR 540 |
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Human Melanoma Primer Library |
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Human NFKappaB Primer Library |
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Human Oncogene Primer Library |
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Mouse Apoptosis Primer Library |
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Mouse Autophagy Primer Library |
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Human Autophagy Primer Library |
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Human Lung Cancer PCR Primer Library |
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Human Apoptosis Primer Library |
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Rat Bdnf Signaling Primer Library |
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| RBDNF-I | Real Time Primers | 1 set | EUR 774 |
Rat Gdnf Signaling Primer Library |
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Mouse Ras Signaling Primer Library |
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Mouse p53 Signaling Primer Library |
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| MTPS-I | Real Time Primers | 1 set | EUR 540 |
Mouse WNT Signaling Primer Library |
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Mouse BMP Signaling Primer Library |
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Human Homeobox Gene Primer Library |
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Human BMP Signaling Primer Library |
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Human Cell Adhesion Primer Library |
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Human Ras Signaling Primer Library |
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Human Schwann Cell PCR Primer Library |
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Human TNF Signaling Primer Library |
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Human p53 Signaling Primer Library |
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Human WNT Signaling Primer Library |
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Mouse Gdnf Signaling Primer Library |
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Mouse Tumor Immunity Primer Library |
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Mouse Bdnf Signaling Primer Library |
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Mouse CREB Signaling Primer Library |
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Mouse EGFR Signaling Primer Library |
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Human EGFR Signaling Primer Library |
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Human GDNF Signaling Primer Library |
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Human IL17 Signaling Primer Library |
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Human BDNF Signaling Primer Library |
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Human PDGF Signaling Primer Library |
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Human SMAD Signaling Primer Library |
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Human Tumor Immunity Primer Library |
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| HTIMU-I | Real Time Primers | 1 set | EUR 657.6 |
Human VEGF Signaling Primer Library |
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Rat Oxidative Stress Primer Library |
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Mouse Hippo Signaling Primer Library |
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Mouse Notch Signaling Primer Library |
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Mouse TGF Beta Signaling Primer Library |
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Mouse Angiogenesis Primer Library |
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| MAPL-I | Real Time Primers | 1 set | EUR 540 |
Mouse Adipogenesis Primer Library |
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Mouse Jak/Stat Signaling Primer Library |
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Human Hippo Signaling Primer Pathway |
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Human Heat Shock Protein Primer Library |
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Human Adipogenesis Primer Library |
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Human Angiogenesis Primer Library |
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Human Cancer Driver Gene I Primer Library |
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Human Cancer Driver Gene II Primer Library |
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Human JAK/STAT Signaling Primer Library |
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Human Osteogenesis Primer Library |
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Human Prostate Cancer Primer Library |
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Human Cytoskeleton Primer Library |
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Human TGF Beta Signaling Primer Library |
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Mouse Oxidative Stress Primer Library |
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Human Drug Transporter I Primer Library |
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Human Endothelial Cell Primer Library |
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Human Oxidative Stress Primer Library |
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| HOSL-I | Real Time Primers | 1 set | EUR 540 |
Rat Glucose Metabolism Primer Library |
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Rat Integrin Signaling Primer Library |
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Mouse Hypoxia Signaling Primer Library |
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Mouse PI3K-AKT Signaling Primer Library |
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Mouse DNA Damage Signaling Primer Library |
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Mouse MAP Kinase Signaling Primer Library |
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Human Hypoxia Signaling Primer Library |
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Human Insulin Signaling Primer Library |
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Human PI3K-AKT Signaling Primer Library |
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Human Alzheimer's Disease Primer Library |
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Human Parkinson's Disease Primer Library |
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Mouse Glucose Metabolism Primer Library |
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Mouse Integrin Signaling Primer Library |
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Human Glucose Metabolism Primer Library |
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Human Hedgehog Signaling Primer Library |
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Human Integrin Signaling Primer Library |
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Human Androgen Signaling Primer Library |
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Human Atherosclerosis Primer Library |
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Rat Ubiquitin Metabolism Primer Library |
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Mouse Adherin - Tight Junction Primer Library |
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Human FGF Receptor Signaling Primer Library |
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Human Adherin - Tight Junction Primer Library |
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Human Ligand Gated Ion Channels Primer Library |
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Human NK Cell Mediated Toxicity Primer Library |
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Human Nitric Oxide Signaling Primer Library |
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Human Rho GTPase-Signaling Primer Library |
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Human Signal Transduction Primer Library |
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Human Breast Cancer Signaling Primer Library |
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Human Chromatin Regulation Primer Library |
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Mouse T-Cell Receptor Signaling Primer Library |
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Human Focal Adhesion Signaling Primer Library |
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| HFCA-I | Real Time Primers | 1 set | EUR 657.6 |
Human B-Cell Receptor Signaling Primer Library |
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| HBCR-I | Real Time Primers | 1 set | EUR 657.6 |
Human T-Cell Receptor Signaling Primer Library |
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Mouse Tumor Invasion/Metastasis Primer Library |
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| MTIM-I | Real Time Primers | 1 set | EUR 540 |
Human Fatty Acid and Lipid Metabolism Primer Library |
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| HFLM-I | Real Time Primers | 1 set | EUR 657.6 |
Human Interferon Type I Signaling Primer Library |
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| HIFN-I | Real Time Primers | 1 set | EUR 657.6 |
Human Interferon Type II Signaling Primer Library |
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| HIFN-II | Real Time Primers | 1 set | EUR 657.6 |
Human Tumor Invasion/Metastasis Primer Library |
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| HTIM-I | Real Time Primers | 1 set | EUR 540 |
Human T-Cell Anergy/Immune Tolerance Primer Library |
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Human Th1 and Th2 Cell Differentiation Primer Library |
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| HTHD-I | Real Time Primers | 1 set | EUR 657.6 |
Mouse Toll-like Receptor Signaling Primer Library |
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| newitem | Real Time Primers | 1 set | EUR 657.6 |
Human Toll-like Receptor Signaling Primer Library |
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Rat Neurotransmitter Signaling Primer Library |
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Mouse Cytokine and Chemokine Receptor Primer Library |
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Mouse Myeloid Derived Suppressor Cell Primer Library |
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Human Actin Cytoskeleton Signaling Primer Library |
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Human Adaptive & Innate Immune Response Primer Library |
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| HAIIR-I | Real Time Primers | 1 set | EUR 657.6 |
Human Cytokine and Chemokine Receptor Primer Library |
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| HCCR-I | Real Time Primers | 1 set | EUR 774 |
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.