Ion Torrent Platform Research Articles

Enhanced microbial diversity in the saliva microbiome induced by short-term probiotic intake revealed by 16S rRNA sequencing on the IonTorrent PGM platform

Microbial communities populating several human body habitats are important determinants of human health. Cultivation-free community-wide approaches like bacterial 16S rRNA sequencing recently revolutionized the study of such human-associated microbial diversity, and the continuously decreasing cost/throughput ratio of current sequencing platforms is further enhancing the availability and effectiveness of microbiome research. The IonTorrent PGM platform is among the latest available commercial high-throughput sequencing tools, but it is just starting to be used for 16S rRNA surveys with only episodic assessments of its performance. We present here the first IonTorrent profiling of the human saliva microbiome collected from 12 healthy individuals. In this cohort, a subset of the volunteers was asked to assume a probiotic product, in order to investigate its impact on the composition and the structure of the saliva microbiome. Analysis of the generated dataset suggests the suitability of the IonTorrent platform for 16S rRNA surveys, even though some platform-specific choices are required to optimize the consistency of the obtained bacterial profiles. Interestingly, we found a marked and statistically significant increase of the overall bacterial diversity in the saliva of individuals who received the probiotic product compared to the control group, suggesting a short-term effect of probiotic product administration on oral microbiome composition.

Next Generation MUT-MAP, a High-Sensitivity High-Throughput Microfluidics Chip-Based Mutation Analysis Panel

Molecular profiling of tumor tissue to detect alterations, such as oncogenic mutations, plays a vital role in determining treatment options in oncology. Hence, there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations. Although commercial assays are available to detect genetic alterations in single genes, only a limited amount of tissue is often available from patients, requiring multiplexing to allow for simultaneous detection of mutations in many genes using low DNA input. Even though next-generation sequencing (NGS) platforms provide powerful tools for this purpose, they face challenges such as high cost, large DNA input requirement, complex data analysis, and long turnaround times, limiting their use in clinical settings. We report the development of the next generation mutation multi-analyte panel (MUT-MAP), a high-throughput microfluidic, panel for detecting 120 somatic mutations across eleven genes of therapeutic interest (AKT1, BRAF, EGFR, FGFR3, FLT3, HRAS, KIT, KRAS, MET, NRAS, and PIK3CA) using allele-specific PCR (AS-PCR) and Taqman technology. This mutation panel requires as little as 2 ng of high quality DNA from fresh frozen or 100 ng of DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Mutation calls, including an automated data analysis process, have been implemented to run 88 samples per day. Validation of this platform using plasmids showed robust signal and low cross-reactivity in all of the newly added assays and mutation calls in cell line samples were found to be consistent with the Catalogue of Somatic Mutations in Cancer (COSMIC) database allowing for direct comparison of our platform to Sanger sequencing. High correlation with NGS when compared to the SuraSeq500 panel run on the Ion Torrent platform in a FFPE dilution experiment showed assay sensitivity down to 0.45%. This multiplexed mutation panel is a valuable tool for high-throughput biomarker discovery in personalized medicine and cancer drug development.

ChildSeq-RNA: A Next-Generation Sequencing-Based Diagnostic Assay to Identify Known Fusion Transcripts in Childhood Sarcomas

Childhood sarcomas can be extremely difficult to accurately diagnose on the basis of morphological characteristics alone. Ancillary methods, such as RT-PCR or fluorescence in situ hybridization, to detect pathognomonic gene fusions can help to distinguish these tumors. Two major deficiencies of these assays are their inability to identify gene fusions at nucleotide resolution or to detect multiple gene fusions simultaneously. We developed a next-generation sequencing-based assay designated ChildSeq-RNA that uses the Ion Torrent platform to screen for EWSR1-FLI1 and EWSR1-ERG, PAX3-FOXO1 and PAX7-FOXO1, EWSR1-WT1, and ETV6-NTRK3 fusions of Ewing sarcoma (ES), alveolar rhabdomyosarcoma, desmoplastic small round cell tumor, and congenital fibrosarcoma, respectively. To rapidly analyze resulting data, we codeveloped a bioinformatics tool, termed ChildDecode, that operates on a scalable, cloud-computing platform. Total RNA from four ES cell lines plus 33 clinical samples representing ES, alveolar rhabdomyosarcoma, desmoplastic small round cell tumor, and congenital fibrosarcoma tumors was subjected to ChildSeq-RNA. This accurately identified corresponding gene fusions in each tumor type, with no examples of false positive fusion detection in this proof-of-concept study. Comparison with previous RT-PCR findings demonstrated high sensitivity (96.4%; 95% CI, 82.3%-99.4%) and specificity (100%; 95% CI, 56.6%-100%) of ChildSeq-RNA to detect gene fusions. Herein, we propose ChildSeq-RNA as a novel tool to detect gene fusions in childhood sarcomas at single-nucleotide resolution.

Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2–P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352–356 (P7–P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7–P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of “designer serpins” with novel reactivity and/or specificity.

Semiconductor-based DNA sequencing of histone modification states

The recent development of a semiconductor-based, non-optical DNA sequencing technology promises scalable, low-cost and rapid sequence data production. The technology has previously been applied mainly to genomic sequencing and targeted re-sequencing. Here we demonstrate the utility of Ion Torrent semiconductor-based sequencing for sensitive, efficient and rapid chromatin immunoprecipitation followed by sequencing (ChIP-seq) through the application of sample preparation methods that are optimized for ChIP-seq on the Ion Torrent platform. We leverage this method for epigenetic profiling of tumour tissues.

Next-generation sequencing-based multigene mutational screening for acute myeloid leukemia using MiSeq: applicability for diagnostics and disease monitoring

Routine molecular testing in acute myeloid leukemia involves screening several genes of therapeutic and prognostic significance for mutations. A comprehensive analysis using single-gene assays requires large amounts of DNA, is cumbersome and timely consolidation of results for clinical reporting is challenging. High throughput, next-generation sequencing platforms widely used in research have not been tested vigorously for clinical application. Here we describe the clinical application of MiSeq, a next-generation sequencing platform to screen mutational hotspots in 54 cancer-related genes including genes relevant in acute myeloid leukemia (NRAS, KRAS, FLT3, NPM1, DNMT3A, IDH1/2, JAK2, KIT and EZH2). We sequenced 63 samples from patients with acute myeloid leukemia/myelodysplastic syndrome using MiSeq and compared the results with those obtained using another next-generation sequencing platform, Ion-Torrent Personal Genome Machine and other conventional testing platforms. MiSeq detected a total of 100 single nucleotide variants and 23 NPM1 insertions that were confirmed by Ion Torrent or conventional platforms, indicating complete concordance. FLT3-internal tandem duplications (n=10) were not detected; however, re-analysis of the MiSeq output by Pindel, an indel detection algorithm, did detect them. Dilution studies of cancer cell-line DNA showed that the quantitative accuracy of mutation detection was up to an allelic frequency of 1.5% with a high level of inter- and intra-run assay reproducibility, suggesting potential utility for monitoring response to therapy, clonal heterogeneity and evolution. Examples demonstrating the advantages of MiSeq over conventional platforms for disease monitoring are provided. Easy work-flow, high throughput multiplexing capability, 4-day turnaround time and simultaneous assessment of routinely tested and emerging markers make MiSeq highly applicable for clinical molecular testing in acute myeloid leukemia.

Application of Genotyping-by-Sequencing on Semiconductor Sequencing Platforms: A Comparison of Genetic and Reference-Based Marker Ordering in Barley

The rapid development of next-generation sequencing platforms has enabled the use of sequencing for routine genotyping across a range of genetics studies and breeding applications. Genotyping-by-sequencing (GBS), a low-cost, reduced representation sequencing method, is becoming a common approach for whole-genome marker profiling in many species. With quickly developing sequencing technologies, adapting current GBS methodologies to new platforms will leverage these advancements for future studies. To test new semiconductor sequencing platforms for GBS, we genotyped a barley recombinant inbred line (RIL) population. Based on a previous GBS approach, we designed bar code and adapter sets for the Ion Torrent platforms. Four sets of 24-plex libraries were constructed consisting of 94 RILs and the two parents and sequenced on two Ion platforms. In parallel, a 96-plex library of the same RILs was sequenced on the Illumina HiSeq 2000. We applied two different computational pipelines to analyze sequencing data; the reference-independent TASSEL pipeline and a reference-based pipeline using SAMtools. Sequence contigs positioned on the integrated physical and genetic map were used for read mapping and variant calling. We found high agreement in genotype calls between the different platforms and high concordance between genetic and reference-based marker order. There was, however, paucity in the number of SNP that were jointly discovered by the different pipelines indicating a strong effect of alignment and filtering parameters on SNP discovery. We show the utility of the current barley genome assembly as a framework for developing very low-cost genetic maps, facilitating high resolution genetic mapping and negating the need for developing de novo genetic maps for future studies in barley. Through demonstration of GBS on semiconductor sequencing platforms, we conclude that the GBS approach is amenable to a range of platforms and can easily be modified as new sequencing technologies, analysis tools and genomic resources develop.

Validation of next-generation sequencing technologies in genetic diagnosis of dementia

Identification of a specific genetic cause of early onset dementia (EOD) is important but can be difficult because of pleiotropy, locus heterogeneity and accessibility of gene tests. Here we assess the use of next generation sequencing (NGS) technologies as a quick, accurate and cost effective method to determine genetic diagnosis in EOD. We developed gene panel based technologies to assess 16 genes known to harbour mutations causal of dementia and combined these with PCR based assessments of the C9orf72 hexanucleotide repeat expansion and the octapeptide repeat region of PRNP. In a blinded study of 95 samples we show very high sensitivity and specificity are achievable using either Ion Torrent or MiSeq sequencing platforms. Modifications to the gene panel permit accurate detection of structural variation in APP. In 2/10 samples which had been selected because they possess a variant of uncertain significance the new technology discovered a causal mutation in genes not previously sequenced. A large proportion (23/85) of samples showed genetic variants of uncertain significance in addition to known mutations. The MRC Dementia Gene Panel and similar technologies are likely to be transformational in EOD diagnosis with a significant impact on the proportion of patients in whom a genetic cause is identified.

A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.

Targeted next-generation sequencing (NGS) of circulating tumor cells (CTCs) in hormone-sensitive prostate cancer (HSPC).

11040 Background: Recently a succession of new hormonal therapies has emerged, highlighting the need for biomarkers to guide the management of HSPC. Biomarker development in HSPC has been hampered by the absence of primary tumor tissue in men who undergo radiation or present with metastatic disease. CTCs can address this challenge by providing real-time cancer tissue for biomarker analysis in HSPC. To test this approach we conducted a pilot of CTC capture and targeted NGS in HSPC. Methods: Under IRB approval, blood samples from patients with HSPC were labeled with EpCAM ferrofluid and placed into the LiquidBiopsy platform (Cynvenio Biosystems, Inc.), an immunoaffinity-based microfluidic device tailored to query genomic events. CTCs were identified by CK, CD45 and DAPI expression. A matched WBC pellet served as a control representing germline sequence. Amplicon libraries were generated using Life Technologies AmpliSeq 2.0 and sequenced on an Ion Torrent platform. Somatic single nucleotide variants (SNV) present in CTCs but not in WBC were identified. Results: CTCs were detected in all 8 patients with HSPC (CTC median 64.5, range 17-217). Germline variants were consistently detected in matched CTC and WBC samples. Significant SNVs (occurring in > 1% of DNA in a sample) were found in 4 of 8 CTC samples (range 1-5 SNVs/sample, frequency 1.2%-11.9% with 620X-14,422X coverage). Notably, 3 patients had biochemical recurrence only (no clinical metastases) yet still yielded CTCs associated with SNVs in KIT, APC, RET, SMAD4 and PTEN. One patient who had untreated metastatic disease had the highest number of CTCs which harbored 4 SNVs. Conclusions: This pilot demonstrates the feasibility of using CTCs as real-time disease relevant substrate for NGS to identify personalized genomic targets in HSPC. A high number of CTCs were detectable in all patients and CTC germline variants correlated with matched WBC controls. Encouragingly, even with a relatively narrow, primary tumor-derived AmpliSeq platform, cancer relevant SNVs were detected in half of the patients including those with only biochemical recurrence, making targeted NGS of CTCs a promising approach for biomarker discovery and validation in HSPC.

Frequent mutations in MLH1, MET, KIT, PDGFRA, and PIK3CA genes in human gastrointestinal stromal tumors.

10544 Background: Identifying mutations in individual tumor is critical to improve the efficacy of cancer therapy by matching targeted drugs to specific mutations. Gastrointestinal stromal tumor (GIST) is a stromal or mesenchymal subepithelial neoplasm affecting the gastrointestinal tract and it frequently contains an activating mutation in either the KIT or platelet-derived growth factor A (PDGFRA) gene. Although GIST is highly responsive to several selective tyrosine kinase inhibitors, combined use of inhibitors targeting other mutations is needed to further prolong survival. Methods: In this study, we aim to screen and identify genetic mutations in GIST for targeted therapy using the new Ion Torrent next-generation sequencing platform. Results: By utilizing the Ion Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes from DNA extracted from formalin-fixed and paraffin-embedded (FFPE) samples of 125 human gastrointestinal stromal tumors, set up stringent parameters for reliable variant calling by filtering out potential raw base calling errors, and identified frequent mutations in PDGFRA, KIT, APC, MLH1, MET, RET, and PIK3CA. Moreover, frequent missense mutations were detected in MLH1 (12%), MET (16.8%), KIT (51.2%), and PIK3CA (41.6%) genes. The mutations in MLH1 gene were frequently detected in the exon 14, while MET gene was frequently mutated in the exon 2. In addition, we identified missense mutations in other genes, including FLT3, KRAS, PDGFRA, and STK11, at lower frequencies. Conclusions: This study demonstrates the utility of using Ion Torrent sequencing to efficiently identify human cancer mutations. With the finding of the frequent mutations in MLH1 and MET genes, in addition to KIT, PDGFRA and PIK3CA, in GISTs, this study provides a molecular basis for clinically developing new drugs targeting these gene mutations for GIST therapy.

Abstract 3509: Complementary analysis of LKB1/STK11 mutation and protein expression status using next-generation sequencing, Sanger sequencing and immunohistochemistry.

Abstract Introduction: LKB1 (liver kinase B1)/STK11 (serine-threonine kinase 11) is a tumor suppressor that encodes a serine/threonine kinase which negatively regulates the mTOR (mammalian target of rapamycin) signaling pathway. Somatic mutations in LKB1 occur most notably in lung adenocarcinoma, cervical cancers and melanoma. Inactivation of LKB1 is caused by point mutations, homozygous deletions or promoter methylation. LKB1 mutation status is a predictive marker for responsiveness to both MEK and PI3K inhibitors. We have developed next-generation sequencing (NGS), Sanger sequencing and immunohistochemistry (IHC) assays to assess LKB1 mutation and protein expression status in a set of FFPE patient specimens. Methods: For NGS, we used the Ion Torrent platform to sequence a 4.4kb region of interest (ROI) around LKB1. A combination of TorrentSuite and in-house tools were used for alignment, variant calling and prioritization of variants. Sanger sequencing was used to validate the presence and absence of variants with a frequency of at least 10% that were identified by NGS. An IHC assay was developed using a commercially available antibody. These assays were validated using a combination of cell lines, a genetically engineered mouse model and FFPE tissue for specificity, sensitivity, reproducibility and concordance. Results: 35 patient FFPE tissues from a variety of tumors including cervix, endometrium, pancreas, skin and lung were evaluated for LKB1 mutation status using NGS and IHC methods. About 96% of the coding region of LKB1 had sufficient sequencing coverage (read depth >500x) to reliably call low frequency somatic variants. Analytical validation using cell line dilutions demonstrated that our NGS assay is able to detect single base changes with frequencies as low as 2-5%, and small indels with frequencies as low as 5-10%. We identified 9 coding and splice site variants in our preliminary analysis of 18/35 of the FFPE specimens. We compared the variants in 7 of these FFPE samples to the variants identified in these samples by Sanger sequencing, and found 100% concordance between the variant calls, for NGS variants with frequencies greater than 20%. Sanger sequencing in the remaining FFPE samples is ongoing, and lower frequency NGS variants will be confirmed by other methods. We compared the validated NGS variants with the IHC results to examine the correlation of sequencing results with protein expression analysis. In our preliminary analysis of the first 18 FFPE samples, all of the samples with an IHC score of zero or one (IHC negative) had either a nonsense (n=1) or missense (n=4) variant in STK11. Conclusions: Our data suggest that accurate assessment of LKB1 status may require complementary methods including NGS and IHC methods. Citation Format: Kimberly Pelak, Jennifer Wright, Zhenyu Yan, Agus Darwanto, Weihua Liu, Peng Fang, Jin Li, Sabita Sankar, Chad Galderisi. Complementary analysis of LKB1/STK11 mutation and protein expression status using next-generation sequencing, Sanger sequencing and immunohistochemistry. [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 3509. doi:10.1158/1538-7445.AM2013-3509

Results from Ion AmpiSeq Cancer Panel in 200 cases of gastric and gastroesophageal junction cancer.

39 Background: We used the Ion AmpiSeq Cancer panel which contains multiplex PCR primers covering 739 potential cancer- related mutations in 46 genes to profile 200 gastric and gastroesophageal cancers. Methods: The assay requires only 10ng of genomic DNA isolated from formalin-fixed paraffin-embedded (FFPE) archival tissue blocks. Samples were retrieved from biopsies from either the primary or metastatic lesion. All samples generated usable DNA and the resultant amplicons were sequenced on the Ion Torrent PGM platform to achieve in depth coverage of potential mutations. Results: Mutations in the tumour suppressor gene TP53 (39.5%) are most commonly identified in our cohort which is consistent with known mutation profiling of gastric carcinoma. We have also identified mutations in MET (8%), PIK3CA (7.5%), KRAS (4%), BRAF (4%) in addition to other known oncogenes. Lastly, We identified two cases with hotspot mutations in IDH1 (R132H and R132C), hotspot mutation traditionally associated with glioma and acute leukemia, which suggest novel role(s) of this mutant protein in the pathogenic progression of this cancer. Conclusions: These findings might predict response to targeted therapeutic agents or have prognostic implications. This study highlights the potential of focused profiling of cancer related genes using the Ion Torrent platform and its advantage of utilizing small amount of DNA from archival pathology specimens.

Editors’ pick: re-‘colon’-ization of healthy microbiota after recurrent C. difficile infection

Editors’ pick: re-‘colon’-ization of healthy microbiota after recurrent C. difficile infection

Characterizing and measuring bias in sequence data

BackgroundDNA sequencing technologies deviate from the ideal uniform distribution of reads. These biases impair scientific and medical applications. Accordingly, we have developed computational methods for discovering, describing and measuring bias.ResultsWe applied these methods to the Illumina, Ion Torrent, Pacific Biosciences and Complete Genomics sequencing platforms, using data from human and from a set of microbes with diverse base compositions. As in previous work, library construction conditions significantly influence sequencing bias. Pacific Biosciences coverage levels are the least biased, followed by Illumina, although all technologies exhibit error-rate biases in high- and low-GC regions and at long homopolymer runs. The GC-rich regions prone to low coverage include a number of human promoters, so we therefore catalog 1,000 that were exceptionally resistant to sequencing. Our results indicate that combining data from two technologies can reduce coverage bias if the biases in the component technologies are complementary and of similar magnitude. Analysis of Illumina data representing 120-fold coverage of a well-studied human sample reveals that 0.20% of the autosomal genome was covered at less than 10% of the genome-wide average. Excluding locations that were similar to known bias motifs or likely due to sample-reference variations left only 0.045% of the autosomal genome with unexplained poor coverage.ConclusionsThe assays presented in this paper provide a comprehensive view of sequencing bias, which can be used to drive laboratory improvements and to monitor production processes. Development guided by these assays should result in improved genome assemblies and better coverage of biologically important loci.

Tips and tricks for the assembly of a Corynebacterium pseudotuberculosis genome using a semiconductor sequencer

New sequencing platforms have enabled rapid decoding of complete prokaryotic genomes at relatively low cost. The Ion Torrent platform is an example of these technologies, characterized by lower coverage, generating challenges for the genome assembly. One particular problem is the lack of genomes that enable reference-based assembly, such as the one used in the present study, Corynebacterium pseudotuberculosis biovar equi, which causes high economic losses in the US equine industry. The quality treatment strategy incorporated into the assembly pipeline enabled a 16-fold greater use of the sequencing data obtained compared with traditional quality filter approaches. Data preprocessing prior to the de novo assembly enabled the use of known methodologies in the next-generation sequencing data assembly. Moreover, manual curation was proved to be essential for ensuring a quality assembly, which was validated by comparative genomics with other species of the genus Corynebacterium. The present study presents a modus operandi that enables a greater and better use of data obtained from semiconductor sequencing for obtaining the complete genome from a prokaryotic microorganism, C. pseudotuberculosis, which is not a traditional biological model such as Escherichia coli.

Next-Generation Sequencing of the BCR-ABL1 Kinase Domain and Neighboring Domains Associated with Therapy Resistance.

AbstractAbstract 2549 Background:BCR-ABL1 mutation testing is recommended for CML and Ph+ ALL patients who fail first line tyrosine kinase inhibitor (TKI) therapy or who have a suboptimal response to therapy. BCR-ABL1 mutations in the kinase domain (KD) of ABL1 account for at least 40–50% of all TKI resistant cases. Rare mutations such as E123Q and T212R in the regulatory domain of ABL upstream of the kinase domain have also been reported to lead to resistance to imatinib. The current gold standard for BCR-ABL1 mutation detection is Sanger sequencing, which has an analytical sensitivity of ∼10–30%. Based on recent findings that mass spectrometry can identify low level BCR-ABL1 mutations that confer clinical resistance in patients sooner than Sanger sequencing, it is likely useful to have a significantly more sensitive BCR-ABL1 test than Sanger sequencing. Although commercial NGS cancer panels have included ABL1 in the region of interest, ABL1 resistance mutations should be sequenced from BCR-ABL1 fusion transcripts instead of being sequenced from genomic DNA as in the commercial panels. Here we developed a fusion transcript based BCR-ABL1 mutation assay on the scalable and cost-effective Ion Torrent platform that has 1–5% sensitivity and comprehensive coverage of the kinase domain, regulatory domain, and the SH2/SH3 domains. The assay was designed to detect both the major and minor BCR-ABL1 fusion gene products and can also detect the micro BCR-ABL1 fusion product accounting for over 99% of all CML and Ph+ ALL patients. Methods:RT and long range PCR was performed to amplify BCR-ABL1 e1, e13, and e14 fusion transcripts and the PCR products were enzymatically fragmented and ligated with Ion Torrent sequencing adaptors. Size-selected libraries were quantified, pooled, amplified with OneTouch system and sequenced with Ion Torrent PGM. Sequencing data was analyzed with Torrent Suite 2.2 and the associated variant caller with variant frequency cutoff adjusted to 1%. Results:Initial work with cell lines harboring the T315I mutation in both e1 and e14 BCR-ABL1 transcript types diluted into wild type cell line demonstrated that Ion Torrent NGS can detect T315I at least down to 1%. In a set of 17 blinded clinical samples, Ion Torrent NGS not only identified all the mutations found by Sanger sequencing but additionally identified rare imatinib resistant mutations such as K357E (MMD-3) present at 7.6%; this mutation was previously reported in patient CD34+CD38-stem cells (Table 1). This patient also expressed the E255K resistance mutation; in patients, the presence of multiple resistance mutations has been shown to be an important predictor of poor response. [Display omitted] Understanding whether compound mutations are present in cis or in trans may be important in understanding therapy resistance. For patient MMD-9, although both the predominant mutation G250E (79%) and subclone E255V(12.6%) were identified by both Sanger sequencing and NGS, only Ion Torrent was able to show that the two mutations were on different reads, indicating that the mutations are on different alleles. The BCR-ABL1 Ion Torrent based assay reads DNA fragments between 150–200bps in size and can identify cis and trans mutations from individual fragments with this read length, which is not possible by Sanger sequencing. In MMD-9, another low frequency subclone F359C (3%) was detected by only NGS and may have important implications due to its reported sensitivity to dasatinib and not imatinib or nilotinib. Similarly, in MMD-10 the E255V predominant mutation was identified by both NGS and Sanger sequencing, while the F317L mutation with a low frequency of 1.70% was solely detected by NGS. In this patient, a combination of dasatinib and nilotinib treatment may be required to eliminate the dasatinib sensitive E255V dominant clone and F317L nilotinib sensitive subclone. Ion Torrent NGS was also capable of identifying and calling the 35 base pair 475 insertion from 4 samples (MMD-2 and 8–10), only two of which were detected by Sanger sequencing.In our ongoing study, the low-level mutations not detected in Sanger sequencing will be confirmed with analyses on the MiSeq platform and mutation enrichment methods. Additional patient samples representing 30 CML patients will be analyzed and presented. Disclosures:Wong:MolecularMD: Equity Ownership.

BIGrat: a repeat resolver for pyrosequencing-based re-sequencing with Newbler

BackgroundAs more and more reference genome sequences are assembled, it becomes practical to assemble individual genomes from large amount of raw read data based on a reference sequence. However, most available assembly tools are designed for de-novo genome assembly. There is one commercial tool box (Newbler) developed for re-sequencing projects based on the Roche 454 sequencing platform. However, the genome with large repeat regions cannot be well assembled in Newbler.FindingsWe developed a new sequence assembly tool (BIGrat, Beijing Institute of Genomics Re-Assembly Tool) for pyrosequencing-based re-sequencing projects, such as data generated from Roche 454 and IonTorrent platforms. BIGrat improves the output of Newbler when evaluated on genome assemblies including chloroplast, mitochondrial, bacterial, and plant nuclear genomes.ConclusionWe presented a novel sequence assembly tool BIGrat for pyrosequencing-based re-sequencing projects, which can easily be integrated into Newbler pipelines for next-generation sequencing assembly and analysis.

Reflex™: a novel method to sequence barcoded long-range PCR products in a pooled population of hundreds of DNA samples

We are developing novel methods to simultaneously analyze candidate genes or regions from multiple samples in a single experiment rapidly and cost-effectively. One method, we call Reflex, provides an elegant approach to sample preparation for long-range PCR (LR-PCR) amplicons. Current methods use LR-PCR for target enrichment then use random fragmentation of each sample followed by ligation of DNA barcodes before sequencing: this approach is expensive and labour intensive. In the Reflex workflow, we perform LR-PCR on genomic targets using primers that also add a DNA barcode to the LR-PCR product. This allows us to then produce pools of the LR-PCR products of multiple, typically 384, samples. The equimolar pooled population of 384 LR-PCR products are then processed to generate tiled 'daughter' Reflex PCR products across the target, each carrying a copy of the cognate DNA barcode. This is achieved by an intramolecular fold-back between two inverted Reflex sequences, followed by polymerase extension, so copying the DNA barcode. The Reflex daughter PCR products are then equimolar pooled for sequencing where the DNA barcode allows identification of the originating DNA sample within the population pool. We have developed Reflex workflows that are compatible with the Illumina, Ion Torrent and Roche-454 next-generation sequencing platforms and can index multiple pooled populations to sequence thousands of DNA samples in a single run. The workflow is robust and can be performed quickly and cheaply with small amounts of input DNA and with high specificity to discriminate between members of multigene families.

Next generation sequencing for TCR repertoire profiling: Platform‐specific features and correction algorithms

The TCR repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of next generation sequencing applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.