Next-generation Sequencing Run Research Articles

Abstract 2938: Comprehensive genomic profiling of solid tumor patients with the OncoDEEP assay for broad analysis in clinical diagnostics

Abstract Somatic multi-gene analysis by Next Generation Sequencing (NGS) becomes standardly integrated in medical oncology for clinical decision making. However, with the fast-growing number of recommended and required genomic biomarkers, small panels have become vastly insufficient for most tumor types. Comprehensive Genomic Profiling (CGP) is amenable to screen for single nucleotide variants (SNVs) and subtle insertions and deletions (indels) in several hundreds of genes. Moreover, it provides information on copy number variations (CNVs) and gene fusions of the most relevant genes for optimal patient management. Currently, most comprehensive panels also allow for screening of tumor-agnostic genomic biomarkers, including microsatellite instability (MSI), tumor mutation burden (TMB) and homologous recombination deficiency (HRD), which are implemented as prognostic and therapeutic signatures in a wide variety of solid tumors. So far, only a handful of CGP assays have been validated for their diagnostic utility in routine laboratory practice for the care of cancer patients. In this study we report on an extensive multicentric analysis across multiple centers in Belgium and The Netherlands comparing the novel OncoDEEP CGP assay (OncoDNA) with the diagnostically validated TSO500 assay (Illumina). We describe the technical differences between both assays as well as their outcome and shortcomings. Detection of clinically relevant SNVs, indels and CNVs was very similar and differences were often due to assay-specific settings. Similarly, TMB, MSI and HRD data were concordant for most samples but those with scores close to the cut-offs could deviate. For fusion detection, concordance was found for the limited number of 11 clinically-actionable driver genes covered by the OncoDEEP kit. The uniformity of coverage was much higher with the OncoDEEP assay thereby allowing to pool 2- to 3-times more samples per NGS run. For the diagnostic implementation of the OncoDEEP assay we then performed an extensive validation with clinical samples representing a wide variety of solid tumor types, as well as reference samples. All performance metrics passed the validation criteria. In conclusion, the OncoDEEP kit can be used for reliable diagnostic comprehensive profiling of solid tumors, but currently, extensive fusion analysis requires an additional screening method. Citation Format: Ernst-Jan M. Speel, Pieter-Jan Volders, Joni Van der Meulen, Aaron De Cock, Stefanie Vermeire, Jacques Van Huysse, Marie De Barsy, Gabriela Beniuga, Wendy de Leng, Anne Jansen, Sharon Thijssen, Hendrikus Jan Dubbink, Zeliha Ozgur, Wilfred van Ijcken, Brigitte Maes, Guy Froyen. Comprehensive genomic profiling of solid tumor patients with the OncoDEEP assay for broad analysis in clinical diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2938.

Direct next-generation sequencing analysis using endometrial liquid-based cytology specimens for rapid cancer genomic profiling.

Genomic examination of cytology specimens is often performed on cell blocks or conventional smears rather than on liquid-based cytology (LBC) specimens. Since LBC specimens preserve high-quality DNA, cancer genome profiling using next-generation sequencing (NGS) is also attainable from residual LBC specimens. One of the advantages of using LBC specimens for NGS is that it allows direct extraction of DNA from residual specimens, avoiding a sacrifice of smear slides and minimizing genomic profiling processing time. Endometrial LBC specimens were subjected to NGS analysis to validate the practicality of rapid cancer genomic profiling in a pathology laboratory. The extracted DNA was subjected to NGS using a customized cancer gene panel comprising 56 genes and 17 microsatellite regions. The workflow strategy was defined, and the processing time estimated for specimen sampling, cell counting, NGS run, and genome profiling. NGS analysis of most LBC specimens revealed somatic mutations, tumor mutation burden, and microsatellite instability, which were almost identical to those obtained from formalin-fixed paraffin-embedded tissues. The processing time for direct NGS analysis and cancer genomic profiling of the residual LBC specimens was approximately 5 days. The residual LBC specimens collected using endometrial cytology were verified to carry a high tumor fraction for NGS analysis and could serve as an alternate source for rapid molecular classification and diagnosis of endometrial cancers, as a routine process in a pathology laboratory.

Harmonization of Next-Generation Sequencing Procedure in Italian Laboratories: A Multi-Institutional Evaluation of the SiRe® Panel.

Background: Next-generation sequencing (NGS) needs to be validated and standardized to ensure that cancer patients are reliably selected for target treatments. In Italy, NGS is performed in several institutions and harmonization of wet and dry procedures is needed. To this end, a consortium of five different laboratories, covering the most part of the Italian peninsula, was constituted. A narrow gene panel (SiRe®) covering 568 clinically relevant mutations in six different genes (EGFR, KRAS, NRAS, BRAF, cKIT, and PDGFRα) with a predictive role for therapy selection in non-small cell lung cancer (NSCLC), gastrointestinal stromal tumor, colorectal carcinoma (CRC), and melanoma was evaluated in each participating laboratory.Methods: To assess the NGS inter-laboratory concordance, the SiRe® panel, with a related kit and protocol for library preparation, was used in each center to analyze a common set of 20 NSCLC and CRC routine samples. Concordance rate, in terms of mutation detected and relative allelic frequencies, was assessed. Then, each institution prospectively analyzed an additional set of 40 routine samples (for a total of 160 specimens) to assess the reproducibility of the NGS run parameters in each institution.Results: An inter-laboratory agreement of 100% was reached in analyzing the data obtained from the 20 common sample sets; the concordance rate of allelic frequencies distribution was 0.989. The prospective analysis of the run metric parameters obtained by each center locally showed that the analytical performance of the SiRe® panel in the different institutions was highly reproducible.Conclusions: The SiRe® panel represents a robust diagnostic tool to harmonize the NGS procedure in different Italian laboratories.

A DNA pool of FLT3-ITD positive DNA samples can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation - Testing several different ITD sequences and rates, simultaneously

Internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene is one of the most frequent genetic alteration in acute myeloid leukemia (AML), and it is associated with worse clinical outcome. Not only the presence but also the size, localization and the rate of this variant or the presence of multiple ITDs has prognostic information. The traditional PCR based diagnostic methods cannot provide information about all of these parameters in one assay, however the application of next generation sequencing (NGS) technique can be a reliable solution for this diagnostic problem. In order to evaluate the analytical properties of an NGS-based FLT3-ITD detection assay a quality control sample was prepared from DNA of AML patients containing 19 different FLT3-ITD variants identified by NGS. The higher the total read count was in a certain sample of the NGS run, the more ITD variant types could be detected. The maximal sensitivity of FLT3-ITD detection by NGS technique was as low as 0.007% FLT3-ITD/total allele rate, however, below 0.1% rate, the reproducibility of the quantitation was poor (CV > 25%). DNA pools with several FLT3-ITDs can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation testing several different ITD sequences and rates, simultaneously.

Clinical embryo sample tracking and identificationusing the mitochondrial genome

Introduction The mitochondrial genome contains single nucleotide variants (SNVs) that can be used to differentiate individuals. Additionally, mitochondrial DNA (mtDNA) is maternally inherited, providing a novel opportunity for DNA-based confirmation of maternal origin of embryo biopsies during Preimplantation Genetic Testing for Aneuploidy (PGT-A) by Next Generation Sequencing (NGS). Typically biopsies from different patients are processed and sequenced together in parallel as high throughput technologies necessitate sample batching for economical purposes. The mitochondrial genome is sequenced during PGT-A and the depth and breadth of coverage obtained from DOPlify® and the PG-Seq™ kit readily allows SNV analysis, even from a 48 sample NGS run. Here we investigate the clinical performance of RHS’ custom Embryo ID Single Nucleotide Variant (SNV) Panel. Material and methods A large putative panel of mitochondrial SNVs was compiled from published literature and by comparative analysis of PG-Seq™ data in IGV. Any SNVs associated with disease- related markers or in regions of lower depth of coverage using DOPlify™ were excluded. A single panel comprising 48 SNVs was compiled for evaluation. PG-Seq™ typically analyses 48 embryo biopsies in a single NGS run. Therefore, we modelled three separate NGS run scenarios; 4 embryos per patient (ie 12 unique mtDNA genomes), 2 embryos per patient (ie 24 mtDNA genomes), or 1 embryo per patient (ie 48 different mtDNA genomes) and tested performance of the panel in each scenario. For each scenario the appropriate number of mtDNA genomes were randomly selected from a publicly available dataset comprising 377 mtDNA genomes representing the global population (mtDB; http://www.mtdb.igp.uu.se/ ), and the panel's ability to differentiate the samples was assessed. This was replicated 10,000 times for each scenario. Results In the 10,000 simulated cases using the 377 mtDNA genome database and modelling embryos from 12 patients, on average, the SNV panel differentiated 91.5% of embryos. In the simulated cases with embryos from 24 patients, on average, the SNV panel differentiated 84.6% of embryos. In simulated cases with embryos from 48 patients, on average, the SNV panel differentiated 75.0% of embryos. In a clinical setting, if embryo signatures match, the rest of the mtDNA genome can still be used to further differentiate samples. It should be noted that maternally-related patients will share mtDNA genomes. In this case, it will not be possible to distinguish between embryos from these patients using the mitochondrial genome. Conclusions RHS’ PG-Seq™ and the Embryo ID Single Nucleotide Variant (SNV) Panel readily generates a unique embryo signature providing a simple novel identification protocol. RHS’ Embryo ID panel has the potential to improve PGT-A practice by providing a DNA-based confirmation of maternal origin and sibling embryo identification. This could be used for sample tracking within an IVF or genetic service provider laboratory. The discriminatory power of the panel may be improved simply by inclusion of population-specific SNVs. Embryo ID will be incorporated into a future release of PG- Seq™ software.

Comprehensive workflow for PGT by NGS: detection of aneuploidies, monogenic disorders and balanced translocations from a single biopsy

Introduction Next Generation Sequencing (NGS) has entered in reproductive genetics for PGT-A with a great success. However, no solution for other alterations such as monogenic disorders and detection of balanced translocations has been developed. We have developed a complete workflow that can be run from a single biopsy. The workflow combines aneuploidy screening (PGT-A) with the additional analysis of monogenic disorders (PGT-M). For PGT- M, the test includes the direct detection of the mutation (when feasible) and the indirect test by analyzing several polymorphisms, previously selected by bioinformatic analysis, around the mutation. This test can be also applied to patients carrying balanced translocations. Material and methods Amplification and library preparation for PGT-A has been done using Ion Reproseq™ (Thermo Fisher). If its combination with PGT-M is required, an Ampliseq panel (Thermo Fisher) is developed for this gene. This includes the mutation of interest (when possible) and hundreds of selected (by in silico analysis) polymorphisms. These polymorphisms are selected in a way that can be used for any couple requiring this procedure. Both libraries (PGT-A and PGT-M) are combined and sequenced in a single run. The protocol allows the automation of many steps with the Ion Chef™ (Thermo Fisher), reducing hands-on time. For balanced translocations, a similar procedure is followed. Results Up to now, this solution has been developed for more than 60 monogenic disorders and applied tomore than 100 couples. The protocol has been validated with STR analysis and Sanger sequencing. Regarding to balanced translocations, one case study is in progress with promising results. The protocol developed works either manually (with results in 12 hours) and in a semiautomated way. Conclusions We have developed a complete protocol that allows the simultaneous analysis of PGT-A and PGT-M in a single NGS run.

Accurate fidelity analysis of the reverse transcriptase by a modified next-generation sequencing

We evaluated fidelity of various reverse transcriptases (RTs) by a novel method with modified next-generation sequencing (NGS). In the optimized condition, one NGS run could handle cDNA products from multiple cDNA synthesis reactions performed at different conditions. This was achieved using a primer containing not only the tag of 14 randomized bases to label each cDNA molecule but also a tag of five bases to label each reaction condition. With this method, we quantitated the error rates of 44 cDNA synthesis reactions by retroviral RTs or genetically engineered DNA polymerases with RT activity under different conditions. The results indicated that high concentrations of MgCl2, Mn(OCOCH3)2, and dNTP decrease the fidelity and that these effects are more pronounced in reactions using RT from human immunodeficiency virus type 1. This is the first report about a precise fidelity monitoring of various RTs by a direct sequence determination.

P073 Multiplexing a variety of clinicaaximize capacity of ion torrent next generation sequencing

Aim We have established the Ion-torrent based Next Generation Sequencing (NGS) system in our clinical laboratory. The chief use of the combination Ion-ChefTM and Ion-S5 is for high resolution HLA typing. However, the full sequencing capacity of the system is not always reached with the HLA typing workflow. In this study, we validated the practicality of combining library pools of different clinical tests into a single NGS run. Methods HLA library pools of 24–32 patient samples were generated with NXType™ Ion-Torrent NGS for Class I (HLA-A, -B, and -C) and Class II (HLA-DR, HLA-DQ, and HLA-DP). These libraries were run alone and then separately combined with library pools of other clinical NGS-based assays. These included: (1) our lab-generated custom tests (APOL1 genotyping (single gene, three SNPs) and Cholestasis genotyping (15 genes, multiple SNPs and INDELS), and (2) commercial assays (16S-Metagenomics, Thermo Fisher Scientific, Inc.) and Lymphotrack, T and B cell clonality (Invivoscribe). All library pools were run on Ion-Chef™ and Ion-S5. HLA data was analyzed with TypeStream™ software v1.1.0.11. The concentration of HLA library pools was maintained at 100 pM. Importantly, the concentration of library pools of other tests were adjusted between 5 and 20 pM, depending on the test (number of genes and size of amplicons) and the number of samples in the pool. Exported data of APOL1, Cholestasis and 16S-Metagenomics were analyzed with Ion Reporter™ software. Lymphotrack data was exported and analyzed with Lymphotrack data analysis software. Results The number of reads covering HLA genes were slightly reduced when runs were combined with libraries of other tests, however, the results of HLA library pools run alone and combined were 100% concordant. The coverage and read numbers obtained for the other tests were sufficient or exceeded the minimal required. Conclusions To our knowledge, this is the first report showing that multiplexing different assays in a single run of Ion-S5 was achievable. We demonstrated that mixing sequencing samples of different genotyping tests in a single NGS run generated accurate results of each individual test. The findings of this works have a positive impact reducing turnaround times and sequencing costs.

Fifteen microsatellite markers for Herbertia zebrina (Iridaceae): An endangered species from South American grasslands.

Premise of the study:Polymorphic microsatellite loci were developed and used to genotype individuals of Herbertia zebrina (Iridaceae) as a first step for assessment of intraspecific genetic diversity.Methods and Results:Primer pairs for 47 markers were developed: 20 from a microsatellite-enriched library and 27 from a next-generation sequencing run using the Illumina MiSeq platform. Of those, 15 loci were considered successful, of which 12 were polymorphic and three were monomorphic. The primers were tested in 50 individuals from three populations of H. zebrina. Two to 14 alleles per locus were identified, and observed and expected heterozygosity were 0.00–0.95 and 0.18–0.89, respectively. Tests of cross-amplification to evaluate the applicability of these markers showed positive results in one congeneric species, H. darwinii, and in a phylogenetically closely related species, Calydorea crocoides.Conclusions:These microsatellite markers can be used for studies of genetic variation and genetic population structure, as well as to support conservation efforts.

Comprehensive Genetic Diagnostics of Acute Myeloid Leukemia By Next Generation Sequencing

BackgroundAcute Myeloid Leukemia (AML) is the most common acute leukemia in adults with a poor overall prognosis. Although the disease has been extensively characterized on the molecular level, this knowledge is translating only slowly into the clinic, particularly with regard to novel therapeutic concepts. Presumably, this striking imbalance substantially is due to the long time required to complete genetic analyses so that results are not available when treatment has to be initiated. Specifically, cytogenetic examinations to determine the karyotype of the malignant blasts, which has been the most important parameter for risk stratification for more than thirty years, take up to two weeks. Next generation sequencing (NGS) technology essentially catalyzed efforts to dissect the genomic landscape of AML, leading to the identification of a large variety of AML driver genes and distinct molecular risk groups. However, these emerging molecular classes of AML do not cover all patients, implying that karyotyping is not dispensable for AML diagnostics at this point. Here we present an integrated approach to AML diagnostics that incorporates these complementary genetic examinations - focused mutational screening of AML-related genes and karyotyping - in one NGS assay.MethodsWe combined targeted resequencing of DNA and RNA using commercially available panels (TruSigth Myeloid, Illumina and FusionPlex Heme, ArcherDx) to detect AML-associated short sequence variants and gene fusions with low coverage whole genome sequencing for copy number variation analysis. Sequencing was performed on an Illumina MiSeq instrument with a read length of 2x150 bp and a coverage of 3.75 M reads for the TruSight Myeloid panel, 2.25 M reads for the FusionPlex panel and 1.5 M reads for the whole genome library. Variants and fusions were called using the manufacturers' analysis software and a previously published algorithm to identify ITDs (ITD-seek, Au et al., 2016). CNV analysis was performed by comparing read distribution in an AML whole genome library to in silico randomly sampled reads from the reference genome using an in house-developed algorithm.ResultsInitial testing of our approach on leukemia cell lines and peripheral blood leukocytes from healthy donors revealed sensitivities of 2% and 1-25% for the detection of DNA variants and fusions, respectively. Applying stringent filter criteria, we recovered 75% of verified COSMIC variants and 100% of known fusions in undiluted AML samples without false positives. Chromosomal gains and losses were detected with high confidence with a sensitivity of 10%. We were able to reliably distinguish between normal and complex karyotypes, although NGS-karyotyping based on known fusions and CNV-analysis missed some details of highly aberrant karyotypes such as derivative chromosomes and chromosomal translocations that did not involve genes included in the FusionPlex panel. Our preliminary experience on our method in a diagnostic setting confirms high correlation with reference laboratory results and no relevant differences with regard to treatment decisions. Moreover, we find that NGS considerably accelerates genetic diagnostics of AML as the entire workflow from sample to report including three parallel library preparations, sequencing and data analysis can be completed within 5 days. Operational costs amount approximately 1,700 USD (1,500 EUR) per sample with the low throughput equipment used in this work, which is in the range of expenses for currently established AML diagnostics.ConclusionsNGS allows for comprehensive translocation and mutation screening, however, some technical and bioinformatics optimization is required to achieve consistently high sensitivity and specificity for all target genes. CNV analysis of low coverage whole genome sequencing data adds valuable information on numerical chromosomal aberrations, thus allowing construction of a virtual karyotype to substitute for difficult and time-consuming cytogenetics. In summary, we present a reliable, fast and cost-effective strategy to combine molecular and cytogenetics for AML diagnostics in a single NGS run in order to pave the way for a more differentiated clinical management of AML patients in the near future. DisclosuresKiehl:Roche: Consultancy, Other: Travel grants, Speakers Bureau.

Accurate clinical detection of exon copy number variants in a targeted NGS panel using DECoN.

Background: Targeted next generation sequencing (NGS) panels are increasingly being used in clinical genomics to increase capacity, throughput and affordability of gene testing. Identifying whole exon deletions or duplications (termed exon copy number variants, 'exon CNVs') in exon-targeted NGS panels has proved challenging, particularly for single exon CNVs. Methods: We developed a tool for the Detection of Exon Copy Number variants (DECoN), which is optimised for analysis of exon-targeted NGS panels in the clinical setting. We evaluated DECoN performance using 96 samples with independently validated exon CNV data. We performed simulations to evaluate DECoN detection performance of single exon CNVs and to evaluate performance using different coverage levels and sample numbers. Finally, we implemented DECoN in a clinical laboratory that tests BRCA1 and BRCA2 with the TruSight Cancer Panel (TSCP). We used DECoN to analyse 1,919 samples, validating exon CNV detections by multiplex ligation-dependent probe amplification (MLPA). Results: In the evaluation set, DECoN achieved 100% sensitivity and 99% specificity for BRCA exon CNVs, including identification of 8 single exon CNVs. DECoN also identified 14/15 exon CNVs in 8 other genes. Simulations of all possible BRCA single exon CNVs gave a mean sensitivity of 98% for deletions and 95% for duplications. DECoN performance remained excellent with different levels of coverage and sample numbers; sensitivity and specificity was >98% with the typical NGS run parameters. In the clinical pipeline, DECoN automatically analyses pools of 48 samples at a time, taking 24 minutes per pool, on average. DECoN detected 24 BRCA exon CNVs, of which 23 were confirmed by MLPA, giving a false discovery rate of 4%. Specificity was 99.7%. Conclusions: DECoN is a fast, accurate, exon CNV detection tool readily implementable in research and clinical NGS pipelines. It has high sensitivity and specificity and acceptable false discovery rate. DECoN is freely available at www.icr.ac.uk/decon.

Abstract 1389: A unified and streamlined targeted sequencing system for the quantification of DNA mutations and RNA expression markers in lung cancer

Abstract Introduction: The promise of precision medicine relies on the identification of DNA and RNA markers that can individualize patient management. Methods such as next-generation sequencing (NGS) can deduce DNA or RNA sequences, but both types of nucleic acid have not been efficiently and effectively combined into a single NGS workflow. We describe a comprehensive methodology for targeted clinical NGS that reports DNA and RNA variants, provides a streamlined workflow, and accommodates low-input total nucleic acid (TNA) from challenging clinical specimens. Methods: Sample QC was performed using a novel qPCR assay that quantifies discrete populations of amplifiable DNA and RNA from TNA material. PCR-based target enrichment was performed using QuantideX® NGS reagents (Asuragen) and sequenced on the MiSeq® System (Illumina). Bioinformatic analyses were conducted using QuantideX® Reporter (Asuragen), a software suite that directly incorporates pre-analytical QC information into the variant calling. Results: Targeted DNA- and RNA-seq panels were developed that query 54 lung cancer DNA targets and 135 RNA targets, including >100 gene fusions and mRNA expression markers associated with clinical actionability. Gene-specific primers were formulated as multiplex PCR or RT-PCR reactions to independently interrogate DNA or RNA variants, respectively, from TNA or combined into a 189-plex RT-PCR to report multi-categorical nucleic acid variants. Integration of the bioinformatics pipeline and variant caller with wet-lab QC results enhanced mutation call sensitivity at <10% abundance, improved PPV for low-input specimens, and achieved absolute quantification of RNA. The NGS panels were assessed with cell-line and synthetic controls and a residual clinical cohort of 97 NSCLC FFPE specimens. Mutations were accurately detected from as few as 5-10 DNA templates and down to <5% abundance, and RNA translocations could be observed in sub-nanogram quantities of fusion-positive FFPE TNA. The unified DNA/RNA panel identified clinically-relevant DNA and RNA variants and maintained similar coverage uniformity to the separate DNA and RNA panels. Analysis of 61 lung adenocarcinoma and 36 squamous cell carcinoma specimens revealed mutation distributions in driver genes and RNA fusions consistent with the known spectrum of variants from each tumor type as previously reported by TCGA and other groups. Conclusions: QuantideX targeted NGS chemistries can unify the analysis of DNA and RNA markers associated with lung cancer and report SNVs, indels, fusions, and aberrantly expressed mRNA transcripts from a single NGS run. This novel technology offers reliable, accurate and comprehensive molecular characterizations of challenging tumor specimens by integrating wet-bench and dry-bench methods and improving the efficacy of routine laboratory testing. Citation Format: Gary J. Latham, Julie Krosting, Michael Dodge, Robert Zeigler, Liangjing Chen, Jason Plyler, Shobha Gokul, Junya Fujimoto, Vassiliki Papadimitrakopoulou, Ignacio Wistuba, Richard Blidner, Brian Haynes. A unified and streamlined targeted sequencing system for the quantification of DNA mutations and RNA expression markers in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1389.

DNA barcodes from century-old type specimens using next-generation sequencing.

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next-generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century-old type specimens of Lepidoptera by attempting to recover 164-bp and 94-bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories--high (164-bp sequence), medium (94-bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR-based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.

Identification of viruses and viroids by next-generation sequencing and homology-dependent and homology-independent algorithms.

A fast, accurate, and full indexing of viruses and viroids in a sample for the inspection and quarantine services and disease management is desirable but was unrealistic until recently. This article reviews the rapid and exciting recent progress in the use of next-generation sequencing (NGS) technologies for the identification of viruses and viroids in plants. A total of four viroids/viroid-like RNAs and 49 new plant RNA and DNA viruses from 18 known or unassigned virus families have been identified from plants since 2009. A comparison of enrichment strategies reveals that full indexing of RNA and DNA viruses as well as viroids in a plant sample at single-nucleotide resolution is made possible by one NGS run of total small RNAs, followed by data mining with homology-dependent and homology-independent computational algorithms. Major challenges in the application of NGS technologies to pathogen discovery are discussed.

High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging.

BackgroundUnambiguous human leukocyte antigen (HLA) typing is important in transplant matching and disease association studies. High-resolution HLA typing that is not restricted to the peptide-binding region can decrease HLA allele ambiguities. Cost and technology constraints have hampered high-throughput and efficient high resolution unambiguous HLA typing. We have developed a method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency. Unambiguous HLA-A, B, C and DRB1 genotypes can be determined for 96 individuals in a single run of the Illumina MiSeq.ResultsLong-range amplification of full-length HLA genes from four loci was performed in separate polymerase chain reactions (PCR) using primers and PCR conditions that were optimized to reduce co-amplification of other HLA loci. Amplicons from the four HLA loci of each individual were then pooled and subjected to enzymatic library generation. All four loci of an individual were then tagged with one unique index combination. This multi-locus individual tagging (MIT) method combined with NGS enabled the four loci of 96 individuals to be analyzed in a single 500 cycle sequencing paired-end run of the Illumina-MiSeq. The MIT-NGS method generated sequence reads from the four loci were then discriminated using commercially available NGS HLA typing software. Comparison of the MIT-NGS with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method.ConclusionsThe MIT-NGS method enabled accurate, robust and cost effective simultaneous analyses of four HLA loci per sample and produced 6 or 8-digit high-resolution unambiguous phased HLA typing data from 96 individuals in a single NGS run.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-864) contains supplementary material, which is available to authorized users.

P099 : A SINGLE CENTER COMPARISON OF HIGH-RESOLUTION HLA TYPING BY NEXT-GENERATION VERSUS SANGER SEQUENCING

Sanger sequencing has been the gold standard for high resolution HLA typing for years. However, typing ambiguities require additional testing to provide allele level resolution. Recently, the development of next-generation sequencing (NGS) technologies for HLA typing has allowed for significantly improved HLA typing. We compared the relative laboratory cost of HLA typing by Sanger sequencing compared and NGS in our moderate volume laboratory. The cost of 30 HLA typings (HLA-A, B, C, DRB1 and DQB1) was compared between Sanger sequencing and NGS. Sanger sequencing was performed with SeCore HLA Typing kits (Life Technologies). Sanger costs included reagent costs for GSSP (SeCore) and/or SSOP (One Lambda) follow up testing for ambiguity resolution. Samples for NGS were amplified using GenDx primers and libraries prepared using Nextera XT kits (Illumina). Sanger samples were run on ABI 3500 while NGS libraries were run on the MiSeq. Analysis was performed using UType software (Sanger) and NGSEngine software (NGS). The reagent cost of Sanger and NGS were based on primers, library preparation and sequencing reagents for each technology. Technologists’ time was an estimation of hands-on time from post-DNA extraction to sequencing data analysis for each method using mean hourly wage. The data was extrapolated to 30 patients, which we anticipate being a typical NGS run. The data doesn’t account for the 7% repeat Sanger sequencing rate we observe for Sanger sequencing run failures. Overall, the cost of HLA typing for 30 patients (5 loci) using NGS was reduced 49% ($22,435) compared to Sanger. Reagent cost for NGS was reduced by $22,073 (49%) compared to Sanger. Technologist time was reduced 60–70% by utilizing NGS instead of Sanger. Sanger sequencing is the gold standard for high-resolution HLA typing however it has limitations. Heterozygous samples often present a problem due to phase ambiguities that require additional testing. The ability of NGS to perform millions of sequencing reads helps correctly phase each nucleotide thereby reducing ambiguities. Moving high-resolution HLA typing from Sanger to NGS will provide substantial savings via multiplexing and reduced reagent cost. Since the cost of NGS instruments and reagents continues to decrease, savings will continue to grow.

Deep sequencing of phage display libraries to support antibody discovery

The use of Next Generation Sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.

Whole genome deep sequencing from single cells for preimplantation genetic diagnosis

FISH and PCR are two main methodologies being used for preimplantation genetic diagnosis (PGD) in the last two decades. Recent advancements in micro-array technology expand the applications of PGD, particularly for preimplatation genetic screening (PGS) to 24 chromosome analysis. A much more powerful approach, next generation sequencing (NGS), is emerging and may have the potential to replace current methodologies for comprehensive genetic analysis. The purpose of this study was to prove the concept that NGS for whole genome analysis on the amplified products from single cells can be used for chromosome copy number assessment and single gene disorder detection. Proof of concept. The whole genome of single cells from known fibroblast cell lines was amplified using commercially available kits. Libraries from amplified DNA were prepared and shotgun deep sequencing was performed using the Roche 454 (Junior) platform. Reads were assembled and aligned using a publically available sequence (Nature, 2008; 456:60) and were analyzed with BWA-SW algorithm. Copy number variation (CNV) was analyzed with MATLAB SegSeq software. NGS on whole genome amplified products from single cells successfully detected the chromosome permutations in cell lines with chromosomal abnormalities. In the experiments we performed, a single NGS run could generate more than one hundred thousand reads, with an average length of 363bp. Computational analysis of the reads indicated the known genetic mutations could be detected. This work indicates that next generation deep sequencing provides high sensitivity and coverage for single cell comprehensive genetic analysis. Thus proves unequivocally that NGS will be a viable technique for clinical PGD in the near future. The method's limitations that exist today, such as speed and cost, are expected to be eliminated in the coming years.