Sample Barcoding Research Articles

Neurofibromatosis type 1 molecular diagnosis: what can NGS do for you when you have a large gene with loss of function mutations?

Molecular diagnosis of neurofibromatosis type 1 (NF1) is challenging owing to the large size of the tumour suppressor gene NF1, and the lack of mutation hotspots. A somatic alteration of the wild-type NF1 allele is observed in NF1-associated tumours. Genetic heterogeneity in NF1 was confirmed in patients with SPRED1 mutations. Here, we present a targeted next-generation sequencing (NGS) of NF1 and SPRED1 using a multiplex PCR approach (230 amplicons of ∼150 bp) on a PGM sequencer. The chip capacity allowed mixing 48 bar-coded samples in a 4-day workflow. We validated the NGS approach by retrospectively testing 30 NF1-mutated samples, and then prospectively analysed 279 patients in routine diagnosis. On average, 98.5% of all targeted bases were covered by at least 20X and 96% by at least 100X. An NF1 or SPRED1 alteration was found in 246/279 (88%) and 10/279 (4%) patients, respectively. Genotyping throughput was increased over 10 times, as compared with Sanger, with ∼90[euro ] for consumables per sample. Interestingly, our targeted NGS approach also provided quantitative information based on sequencing depth allowing identification of multiexons deletion or duplication. We then addressed the NF1 somatic mutation detection sensitivity in mosaic NF1 patients and tumours.

DNA barcoding of Cameroon samples enhances our knowledge on the distributional limits of putative species of Osteolaemus (African dwarf crocodiles)

Recent genetic and morphological evidence indicates that the African dwarf crocodile (O. tetraspis) is comprised of three highly divergent lineages: O. sp. nov., O. tetraspis and O. osborni. Their putative distributional limits correspond with the Cameroon volcanic line (CVL) and the Congo River basin. In this study, we expanded on previous phylogeographic work by conducting detailed sampling of this crocodile in Cameroon and around the CVL. We tested whether O. tetraspis is the only lineage occurring in Cameroon and whether the CVL represents the distributional limit between O. sp. nov. and O. tetraspis. We collected 65 tissue samples from individuals located throughout Cameroon, and the eastern and western sides of the CVL. We sequenced fragments of two mitochondrial genes (CO1 and 12S rDNA) and one nuclear gene (LDH-A). We found that O. tetraspis extends west beyond the CVL and, thus, this mountain chain does not represent the distributional limit of this lineage. We also found O. osborni in Cameroon. Our findings have important implications for the conservation and management of O. tetraspis lineages.

1020-LBP: A system for high-throughput library preparation allows high-resolution HLA typing using next-generation sequencing

Aim The most widely used platform for HLA typing, capillary electrophoresis-based Sanger sequencing technology, does not scale with the number of regions and samples required in complex study designs when compared to next-generation sequencing (NGS) platforms. Here we describe a high-throughput system under development for high-resolution HLA typing capable of generating libraries for NGS using 192 samples and 24 different target specific assays in less than eight hours in run time and 60 minutes in hands-on time. Methods The new Access Array™ HT System is composed of the IFC Controller AXHT, FC1™ thermal cycler, and the 192.24 Access Array™ IFC. The IFC contains 4,608 chambers of 11 nL volume each where amplification reaction takes place. To test the performance of this new system, primers targeting exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 were used to amplify 192 genomic DNA samples previously typed. Sample barcodes and sequencer-specific tags were added to the amplicons during chip amplification step. After amplification, library was prepared for sequencing. Results Eight 192.24 Access Array™ IFCs were used to prepare libraries for NGS. The first library tested DNA input concentrations of 5, 10, and 20 ng/ μ L DNA. The success rate achieved was of 82.5%, 95.3%, and 95.7% respectively, with 100% concordant typing. Additional libraries prepared using 10 ng/ μ L of DNA resulted in 100% concordant typing for 96% or more of all 2,172 loci tested. These results obtained are the first set of data using the platform under development and will improve with platform and primer design optimization. Conclusions The Access Array™ HT System has proven to be a reliable and efficient system to generate libraries for HLA typing, reaching 100% concordance for over 95% of the loci tested. This system fits the needs of an evolving field where typing a large number of samples accurately, at low cost and short turnaround time, is necessary to effectively test genetically heterogeneous specimens.

115-P: AUTOMATED HIGH THROUGHPUT ISOLATION OF LYMPHOID AND MYELOID CELLS DIRECTLY FROM WHOLE BLOOD USING RoboSep-HT

Aim Process automation is an important requirement for streamlining and standardizing technical procedures, such as cell isolation, in a routine laboratory. We have previously developed RoboSep™, the first instrument to fully automate the cell labeling and separation steps of an immunomagnetic cell isolation procedure. RoboSep™ uses EasySep™ reagents and protocols and can isolate up to 4 individual samples at one time. More recently we have developed a high throughput version of RoboSep™, called RoboSep-HT, which can isolate 16 samples from up to 4 different cell types in about 1 hour. Like RoboSep™, RoboSep-HT uses disposable filter tips to avoid cross contamination between samples and is operated using an intuitive graphical user interface. Sample barcoding is a standard feature. In this study, positive selection of CD3+ and myeloid+ cells directly from whole blood using EasySep™ reagents was performed on the new RoboSep-HT and compared to performance using the classic RoboSep™. Methods 1mL of fresh, 24-hour or 48-hour old whole blood was diluted with an equal volume of 1xRBC lysis buffer and loaded on each instrument. Purity (flow cytometry), recovery (cell counts) and DNA yield (OD) were assessed following isolation using each robot. Replicates were averaged prior to statistical analysis (t-tests). Results There was no significant difference in either purities, cell recoveries or DNA yields when CD3 and myeloid cell isolation was performed on the RoboSep-HT versus the classic RoboSep™. [ Table 1 ] Conclusions RoboSep-HT can be used for the high throughput isolation of multiple cell types and is ideally suited for cell isolation prior to chimerism testing. McQueen: STEMCELL Technologies: Employee. Kellerman: STEMCELL Technologies: Employee. Frehlick: STEMCELL Technologies: Employee. Gromadzki: STEMCELL Technologies: Employee. Poon: STEMCELL Technologies: Employee. Woodside: STEMCELL Technologies: Employee. Thomas: STEMCELL Technologies: Employee.

132-P: DEVELOPMENT OF AN HLA TYPING METHOD FOR THE ION TORRENT PGM NEXT GENERATION SEQUENCING PLATFORM

The Ion Torrent PGM™ next generation sequencing platform allows for the simultaneous sequencing of up to 96 barcoded samples in a single two to four hour run, providing a method to process a large number of gene sequences in significantly less time than traditional sequencing. We are developing an HLA typing method based on the technology that enables read lengths of 400 bases on the PGM™ platform. Two different library preparation workflows have been evaluated. The first approach is based on long-range PCR (LR-PCR) followed by fragmentation and ligation of emulsion PCR adaptor sequences. This method allows coverage of an entire HLA gene with a single long-range PCR reaction for each sample. The basis for the second workflow is a tiled, multiplex, short-range PCR design. The amplicons in this design range in length from 350 - 400 base pairs and cover alternating sections of a gene in two primer pools. This approach offers a workflow that does not include the fragmentation and size selection steps required in the LR-PCR method, saving at least four hours of processing time while producing results equivalent to the LR-PCR workflow. Here we describe the development of a tiling assay that enables full coverage of the HLA-C gene. Approximately 100 primer sets were screened in designing the amplicons. Thirty samples have been sequenced using each method to compare coverage of region, read depth, accuracy, cis/trans ambiguity resolution, and ease of workflow. The current design is comprised of two PCR tubes, each with thirty-five primers, used to generate eighteen tiling amplicons. Each primer pool is designed to amplify every HLA-C gene with known genomic sequence. While the LR-PCR method has simplified primer designs, the short-range PCR approach enables a simplified workflow with equivalent genomic sequence results. The assay designs to date show the feasibility of the PGM™ platform for accurate, high throughput HLA gene sequencing. Bialozynski: Life Technologies: Employee. Dinauer: Life Technologies: Employee. Gifford: Life Technologies: Employee. Zhao: Life Technologies: Employee. Shi: Life Technologies: Employee. Radick: Life Technologies: Employee. Conradson: Life Technologies: Employee.

A survey of rare coding variants in candidate genes in schizophrenia by deep sequencing

The genetic architecture of schizophrenia is likely contributed by both common and rare variants.1 Recent genome-wide studies have revealed that common variants in the major histocompatibility complex (MHC) region, TCF4 and other genes are associated with schizophrenia.1 In addition, rare copy-number variation (CNV) regions in broad regions like 1q21.1, 15q13.3, 15q11.2, 22q111 as well as individual genes such as Neurexin2, 3 have been identified. Unbiased exome or whole genome scanning procedures have the potential to identify novel loci while likely requiring large sample sets to reach a genome-wide significance level. It is possible that the previously identified genes/regions from high-throughput single-nucleotide polymorphisms (SNP) chip genome-wide scanning techniques, in contrast to some ‘classical' candidate genes,4 may harbor rare coding variants that have a role in disease risk. We selected a total of 101 genes from within the 1q21.1, 15q13.3, 22q11 and 15q11.2 regions and a number of other candidate genes, with either a priori knowledge for association with schizophrenia, for example, TCF4/CCDC68, NRXN1, or interesting for drug-discovery efforts, for example, cyclic nucleotide phosphodiesterase genes, and surveyed rare variants in their coding regions through deep sequencing.

Abstract 4215: Rapid, inexpensive and accurate detection of rare variants by targeted sequencing.

Abstract Next generation sequencing technology is quickly becoming the most rapid and accurate method of determining mutation profiles of patient's tumor samples. While whole genome or exome sequencing profiles are the most comprehensive methods for complete exploration of these tumors, these protocols typically require large amounts of DNA, are expensive, don't get deep coverage, and require extensive time for library and sample preparation. Ion Torrent has developed new methodology for looking at 10’s to 1000’s of genomic targets with the Ion AmpliSeq™ technology. The recommended input of 10ng of DNA is suitable for work with FFPE or fine needle biopsy samples, and has been demonstrated on samples with less than 1ng of DNA. In a 10 hour day this protocol can take isolated DNA through the library and sample prep as well sequencing with variant calls, making it an incredibly rapid method for determining sequence variants within targeted regions. There are currently two commercially available cancer-specific amplicon panels: the Comprehensive Cancer Panel (CCP) uses ∼16,000 primer pairs covering the exons of 409 tumor suppressor genes and oncogenes frequently cited and mutated in cancers, and the Cancer Hotspot Panel v2 (CHP2) uses 207 primer pairs in 50 clinically actionable genes to detect over 2800 mutation hotspots. We recently measured the sensitivity of SNP detection levels with the CCP and CHP2 panels on a 90:10 (reference:SNP) admixed sample and were able to detect SNP proportions of 2.0%(p<0.005) with CHP2 and 2.0% (p<0.001) with CCP. The CHP2 amplicons yielded coverages of >2000X for a single sample on a 314 chip and >3000X for 6 barcoded samples on a 318 chip. Comparatively, the average base coverage of the CCP was >280x and over 95% of the CCP amplicons were covered at >50X. We ran the CCP in quadruplicate on 318 chips at two separate facilities and the CHP2 was run barcoded on 318 chips and single samples on 314 chips also in quadruplicate at two separate facilities and all of the chips show very equivalent numbers for SNP detection and coverages. These results demonstrate the consistent nature of targeted sequencing using a next gen platform. Going from isolated DNA to variant calling in as little as 10 hrs, this method is extremely fast and efficient which will be critical in future diagnostic settings. Citation Format: Devin Dressman, Stefanie Nishimura, Charles Scafe, Yutao Fu, Ellen Fanti, Lev Kotler, Vaish Panchapakesa, Wendell Orphe, Sherry Hansen, Nick Hapshe, Carl Fergus, Erin Lagier, Eric Wei, Grace Lui, Reddy Nallapareddy, Xiaohui Chen, Zhitong Liu, Eugeni Namsaraev. Rapid, inexpensive and accurate detection of rare variants by targeted sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4215. doi:10.1158/1538-7445.AM2013-4215

Abstract 3231: Comprehensive profiling of actionab.

Abstract Comprehensive characterization of genetic profiles in clinically actionable genes in each tumor promises to facilitate personalized approaches to cancer treatment. However, development of systematic genetic profiling of cancers in the clinical setting is facing unique technical challenges. More than 90% of tumor samples available for clinical sequencing are formalin-fixed and paraffin-embedded (FFPE). These samples are not only limited by quantities, the DNA quality in these samples is often compromised due to different degree of degradation, cross-links and chemical modifications. Targeted sequencing, in which genomic regions of interests are selectively enriched before sequencing to achieve high coverage and sensitivity, can potentially provide a cost-effective and efficient solution for clinical sequencing. However, very few studies have been reported on the development of methodology that is applicable and robust in analyzing “real-world” clinical FFPE samples. Here we report the development of a novel methodology that integrates microfluidic multiplex PCR-based target enrichment with massively parallel sequencing for high-throughput, high-content targeted sequencing of clinically actionable cancer genes in FFPE tumor tissues. This method enables amplifying 963 regions from 88 common cancer genes in 48 samples and simultaneously sequencing 48 pooled barcoded samples in a single sequencing reaction. Using this method, we sequenced 40 cancer cell lines and a well-defined Latin square study with dilution series of 8 known somatic mutations, to comprehensively assess the sensitivity, specificity, reproducibility in both target enrichment and variant detection. Furthermore, using paired fresh frozen/FFPE tissues obtained from 16 cancer patients, we developed and optimized a FFPE protocol that yields high-quality genetic profiles highly concordant to those derived from parallel fresh frozen samples. To demonstrate the clinical utility of this method, we sequenced a cohort of 73 FFPE tumor samples from endometrial cancer patients. Many clinically actionable mutations were identified in these patients’ samples and confirmed using orthogonal validations. Our results demonstrated the feasibility and clinical utility of our method in clinical sequencing, setting the stage for ultimately translating this method in clinical setting to inform and guide cancer treatment. Citation Format: Yulei Wang, Richard Bourgon, Shan Lu, Yibing Yan, Yinghui Guan, Lisa Ryner, Rajesh Partel, Mark Lackner, Lukas Amler. Comprehensive profiling of actionab. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3231. doi:10.1158/1538-7445.AM2013-3231

Abstract P2-16-03: Creation of a “state-of-the-art” breast cancer data and biobank in Argentina

Abstract Translational cancer research is highly dependent on having large series of cases with high-quality samples that were collected, processed, stored and annotated in a systematic fashion according to international standards, such as the ISBER Best Practices for Biorepositories. Sharing successful strategies between the Baylor College of Medicine (BCM) Cancer Center and Breast Center Buenos Aires (BCBA), we created this Argentinean biobank complete with breast cancer-oriented data, tumor and normal breast tissue and matched blood specimens according to best practices to facilitate international collaborative translational breast cancer research. Objective: Creation of a blood, breast tissue and tumor biobank in Argentina for research purposes with associated epidemiological, pathological, clinical, and follow-up data. Methods: Consented individuals from BCBA are classified by treating physician in four categories: 1) breast cancer; 2) benign breast disease by biopsy; 3) high-risk for breast cancer according to the Gail Model; and 4) healthy controls. Blood is collected at several time points during the breast cancer disease process: pre-surgical, pre-systemic treatment, and, if applicable, in the metastatic setting. Blood products are stored as whole blood, plasma, buffy coat, red blood cell pellet, serum and clot at −80°C or at room temperature in GenPlates. Fresh tissue and tumor sample is collected during surgical or core biopsy proceedings and stored at −80°C and paraffin embedded. Upon enrollment, participants complete an extensive epidemiological and risk factor questionnaire, which is supplemented by medical record abstraction for relevant pathological and clinical data. Participants are also re-contacted once a year for follow-up. The creation of this biobank in 2009 included the preparation and adaptation of the following processes from the Population Science Biorepository and Smith Breast Center Tumor Bank at BCM, including: 1) IRB-approved informed consent documents with specific language relevant to DNA-based research; 2) Epidemiological and risk factor questionnaires (10-page Core module and 6-page Breast module); 3) Blood and tissue collection protocols, sample processing, sample bar coding and participant/sample registration system; and 4) Web-based data management system, specifically designed for this biobank with encryption and data security (details presented in a separate abstract), including role-based access levels personal health information. Conclusion: We started our endeavor in August 2008. The development of the consent forms, two questionnaires, and biobank database prototype took one year at BCM. The translation of consents and questionnaires into Argentinean Spanish took an additional 3 months. Overall the Argentinean IRB approval process, training personnel, and equipment set-up lasted for 14 months. By April 2011, the first Argentinean participant was consented into the biobank. It took nearly 3 years from inception to realization for this biobank; however, the potential benefit to translation breast cancer research is large. The overall value of this biobank will depend on the number of individuals/samples accrued and the number of years of follow-up attained. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-16-03.

FLEXBAR-Flexible Barcode and Adapter Processing for Next-Generation Sequencing Platforms.

Quantitative and systems biology approaches benefit from the unprecedented depth of next-generation sequencing. A typical experiment yields millions of short reads, which oftentimes carry particular sequence tags. These tags may be: (a) specific to the sequencing platform and library construction method (e.g., adapter sequences); (b) have been introduced by experimental design (e.g., sample barcodes); or (c) constitute some biological signal (e.g., splice leader sequences in nematodes). Our software FLEXBAR enables accurate recognition, sorting and trimming of sequence tags with maximal flexibility, based on exact overlap sequence alignment. The software supports data formats from all current sequencing platforms, including color-space reads. FLEXBAR maintains read pairings and processes separate barcode reads on demand. Our software facilitates the fine-grained adjustment of sequence tag detection parameters and search regions. FLEXBAR is a multi-threaded software and combines speed with precision. Even complex read processing scenarios might be executed with a single command line call. We demonstrate the utility of the software in terms of read mapping applications, library demultiplexing and splice leader detection. FLEXBAR and additional information is available for academic use from the website: http://sourceforge.net/projects/flexbar/.

Rapid development of molecular resources for a freshwater mussel,Villosa lienosa(Bivalvia:Unionidae), using an RNA-seq-based approach

Freshwater mussels (Unionidae) are among the most endangered groups of organisms in the world, and their conservation and recovery are priorities throughout North America, especially the southeastern USA. We used a ribonucleic acid sequencing (RNA-seq)-based approach to develop molecular resources for Villosa lienosa, the little spectaclecase. We sequenced barcoded samples (Illumina HiSeq 2000 platform) and assembled (Trans-ABySS) 778,234 contigs (average length = 707.5 base pairs [bp]) from 162 million filtered reads. We identified 23,742 unigene hits against the National Center for Biotechnology Information nonredundant database and 36,582 microsatellites with sufficient flanking sequence for primer design. Microsatellite validation indicated a 36% polymorphic rate (16/44 tested markers) in the tested population (26 individuals; mean = 5 alleles/marker). Analysis of differentially expressed genes between heat-stressed and untreated controls enabled us to identify 604 genes involved in stress-response pathways. Real-time polymerase chain reaction validation of gene-expression results using individual samples confirmed RNA-seq patterns (r = 0.847, p < 0.001). RNA-seq is a powerful tool for rapid development of molecular resources in nonmodel species, and our study is the first large-scale transcriptome project in freshwater mussels. The validated microsatellite set and stress-associated genes are being used in parentage analysis and health-assessment surveys to support mussel conservation.

ColoSeq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing

Lynch syndrome (hereditary nonpolyposis colon cancer) and adenomatous polyposis syndromes frequently have overlapping clinical features. Current approaches for molecular genetic testing are often stepwise, taking a best-candidate gene approach with testing of additional genes if initial results are negative. We report a comprehensive assay called ColoSeq that detects all classes of mutations in Lynch and polyposis syndrome genes using targeted capture and massively parallel next-generation sequencing on the Illumina HiSeq2000 instrument. In blinded specimens and colon cancer cell lines with defined mutations, ColoSeq correctly identified 28/28 (100%) pathogenic mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH, including single nucleotide variants (SNVs), small insertions and deletions, and large copy number variants. There was 100% reproducibility of detection mutation between independent runs. The assay correctly identified 222 of 224 heterozygous SNVs (99.4%) in HapMap samples, demonstrating high sensitivity of calling all variants across each captured gene. Average coverage was greater than 320 reads per base pair when the maximum of 96 index samples with barcodes were pooled. In a specificity study of 19 control patients without cancer from different ethnic backgrounds, we did not find any pathogenic mutations but detected two variants of uncertain significance. ColoSeq offers a powerful, cost-effective means of genetic testing for Lynch and polyposis syndromes that eliminates the need for stepwise testing and multiple follow-up clinical visits.

T Cell-Signaling Network Analysis Reveals Distinct Differences between CD28 and CD2 Costimulation Responses in Various Subsets and in the MAPK Pathway between Resting and Activated Regulatory T Cells

To uncover signaling system differences between T cell stimuli and T cell subsets, phosphorylation status of 18 signaling proteins at six different time points following TCR triggering and CD28/CD2 costimulation was examined in human T cell subsets by phospho-epitope-specific flow cytometry of fluorescent cell barcoded samples, thereby providing a high-resolution signaling map. Compared with effector/memory T cells, naive T cells displayed stronger activation of proximal signaling molecules after TCR triggering alone. Conversely, distal phosphorylation events, like pErk and pS6-ribosomal protein, were stronger in effector/memory subsets. CD28 costimulation specifically induced signaling necessary for proper NF-κB activation, whereas CD2 signaled more strongly to S6-ribosomal protein. Analysis of resting regulatory T cells (rTregs; CD4(+)CD45RA(+)FOXP3(+)) and activated regulatory T cells (actTregs; CD4(+)CD45RA(-)FOXP3(++)) revealed that, although rTregs had low basal, but inducible, Erk activity, actTregs displayed high basal Erk phosphorylation and little or no Akt activation. Interestingly, the use of Mek inhibitors to block Erk activation inhibited activation-dependent FOXP3 upregulation in rTregs, their transition to actTregs, and the resulting increase in suppressive capacity. In summary, our systems approach unraveled distinct differences in signaling elicited by CD28 and CD2 costimulation and between rTregs and actTregs. Blocking rTreg transition to highly suppressive actTregs by Mek inhibitors might have future therapeutic applications.

Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing

The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3-5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5-6 d for array-based enrichment and 3-4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussed.

Genetic diagnosis of Duchenne and Becker muscular dystrophy using next-generation sequencing technology: comprehensive mutational search in a single platform

BackgroundDuchenne muscular dystrophy or Becker muscular dystrophy might be a suitable candidate disease for application of next-generation sequencing in the genetic diagnosis because the complex mutational spectrum and the large...

Validation of a Cost-Efficient Multi-Purpose SNP Panel for Disease Based Research

BackgroundHere we present convergent methodologies using theoretical calculations, empirical assessment on in-house and publicly available datasets as well as in silico simulations, that validate a panel of SNPs for a variety of necessary tasks in human genetics disease research before resources are committed to larger-scale genotyping studies on those samples. While large-scale well-funded human genetic studies routinely have up to a million SNP genotypes, samples in a human genetics laboratory that are not yet part of such studies may be productively utilized in pilot projects or as part of targeted follow-up work though such smaller scale applications require at least some genome-wide genotype data for quality control purposes such as DNA “barcoding” to detect swaps or contamination issues, determining familial relationships between samples and correcting biases due to population effects such as population stratification in pilot studies.Principal FindingsEmpirical performance in classification of relative types for any two given DNA samples (e.g., full siblings, parental, etc) indicated that for outbred populations the panel performs sufficiently to classify relationship in extended families and therefore also for smaller structures such as trios and for twin zygosity testing. Additionally, familial relationships do not significantly diminish the (mean match) probability of sharing SNP genotypes in pedigrees, further indicating the uniqueness of the “barcode.” Simulation using these SNPs for an African American case-control disease association study demonstrated that population stratification, even in complex admixed samples, can be adequately corrected under a range of disease models using the SNP panel.ConclusionThe panel has been validated for use in a variety of human disease genetics research tasks including sample barcoding, relationship verification, population substructure detection and statistical correction. Given the ease of genotyping our specific assay contained herein, this panel represents a useful and economical panel for human geneticists.

48 Next-generation sequencing technology combined with multiplexed barcoded samples of myelodysplastic syndromes reveals an abundance of target gene mutations

48 Next-generation sequencing technology combined with multiplexed barcoded samples of myelodysplastic syndromes reveals an abundance of target gene mutations

Sequence Capture and Next-Generation Resequencing of Multiple Tagged Nucleic Acid Samples for Mutation Screening of Urea Cycle Disorders

Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples. We used sectored 2240-feature medium-density oligonucleotide arrays to capture and enrich a 199-kb genomic target encompassing the complete genomic regions of 3 urea cycle genes, OTC (ornithine carbamoyltransferase), CPS1 (carbamoyl-phosphate synthetase 1, mitochondrial), and NAGS (N-acetylglutamate synthase). We used the Genome Sequencer FLX System (454 Life Sciences) to jointly analyze 4 samples individually tagged with a 6-bp DNA bar code and compared the results with those for an individually sequenced sample. Using a low tiling density of only 1 probe per 91 bp, we obtained strong enrichment of the targeted loci to achieve ≥90% coverage with up to 64% of the sequences covered at a sequencing depth ≥10-fold. We observed a very homogeneous sequence representation of the bar-coded samples, which yielded a >30% increase in the sequence data generated per sample, compared with an individually processed sample. Heterozygous and homozygous disease-associated mutations were correctly detected in all samples. The use of DNA bar codes and the use of sectored oligonucleotide arrays for target enrichment enable parallel, large-scale analysis of complete genomic regions for multiple genes of a disease pathway and for multiple samples simultaneously. This approach thus may provide an efficient tool for comprehensive diagnostic screening of mutations.

Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples

Targeted genomic enrichment followed by next-generation DNA sequencing has dramatically increased efficiency of mutation-discovery efforts. We describe a protocol for genomic enrichment of pooled barcoded samples in a single assay that increases experimental flexibility and efficiency. We screened 770 genes (1.4 megabases) in thirty N-ethyl-N-nitrosourea (ENU)-mutagenized rats and identified known variants at >96% sensitivity as well as new mutations at a false positive rate < 1 in 8 megabases.

Reduction of Non-Insert Sequence Reads by Dimer Eliminator LNA Oligonucleotide for Small RNA Deep Sequencing

Here we describe a method for constructing small RNA libraries for high-throughput sequencing in which we have made a significant improvement to commonly available standard protocols. We added a locked nucleic acid (LNA) oligonucleotide--named dimer eliminator--that is complementary to the adapter-dimer ligation products during the reverse transcription reaction. It reduces adapter-dimers, which often contaminate standard libraries and increase the number of non-insert sequence reads. This simple technology can be used for simultaneous multiplex sequencing of various barcoded samples as well as nonbarcoded small RNA library sequencing. In this study we also evaluated the reproducibility and quantitative design of the eight barcoded tags by comparing the Pearson's correlation values in the expression analysis between each barcoded sample. This method improves the sequencing yield and efficiency, while simplifying library construction, and makes it easier to perform large-scale small RNA analysis under multiple conditions with next-generation sequencers.