Ion Torrent Next-generation Sequencing Research Articles

A new nationwide genomic screening system in Japan for the development of targeted therapies against advanced non-small lung cancers with rare driver mutations.

11007 Background: A variety of targetable driver mutations other than EGFR mutations and ALKfusions occur in 1%-2% of NSCLCs. Efficient screening systems for these rare mutations are necessary for the successful development of targeted therapies. Methods: In February 2013, a new nationwide genomic screening system (LC-SCRUM-Japan) was established in Japan to screen advanced non-squamous NSCLCs without EGFR mutations for primarily ALK/RET/ROS1fusions. In November 2013, this system was amended to further screen for other driver mutations in fusion-negative cancers after the primary screening. Ten nanograms of genomic DNA extracted from biopsy or cytology specimens were subjected to the Ion Torrent AmpliSeq Cancer Hotspot Panel, version 2, and Ion Torrent next-generation sequencing, enabling the simultaneous analysis of ˜2800 hotspot mutations in 50 cancer-related genes. Results: As of January 31, 2014, a total of 158 institutions were participating and 507 patients were enrolled in LC-SCRUM-Japan. Among the...

Comparison of targeted next-generation sequencing with conventional sequencing for predicting the responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy in never-smokers with lung adenocarcinoma

PurposeTo investigate the clinical utility of targeted next-generation sequencing (NGS) for predicting the responsiveness to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy, we compared the efficacy with conventional sequencing in never-smokers with lung adenocarcinoma (NSLAs). Patients and methodsWe obtained DNA from 48 NSLAs who received gefitinib or erlotinib for their recurrent disease after surgery. Sanger sequencing and peptide nucleic acid clamp polymerase chain reaction (PCR) were used to analyze EGFR, KRAS, BRAF, and PIK3CA mutations. We analyzed ALK, RET, and ROS1 rearrangements by fluorescent in situ hybridization or reverse transcriptase-PCR and quantitative real-time PCR. After molecular screening, Ion Torrent NGS was performed in 31 cases harboring only EGFR exon 19 deletions (19DEL), an L858R mutation, or none of the above mutations. ResultsThe 31 samples were divided into four groups: (1) responders to EGFR-TKIs with only 19DEL or L858R (n=15); (2) primary resistance to EGFR-TKI with only 19DEL or L858R (n=4); (3) primary resistance to EGFR-TKI without any mutations (n=8); (4) responders to EGFR-TKI without any mutations (n=4). With NGS, all conventionally detected mutations were confirmed except for one L858R in group 2. Additional uncovered predictive mutations with NGS included one PIK3CA E542K in group 2, two KRAS (G12V and G12D), one PIK3CA E542K, one concomitant PIK3CA and EGFR L858R in group 3, and one EGFR 19DEL in group 4. ConclusionsTargeted NGS provided a more accurate and clinically useful molecular classification of NSLAs. It may improve the efficacy of EGFR-TKI therapy in lung cancer.

Ion Torrent next-generation sequencing reveals the complete mitochondrial genome of black and reddish morphs of the Coral Trout Plectropomus leopardus

Using Ion Torrent next-generation sequencing (NGS) technology, we sequenced the complete mitochondrial genome (mitogenome) of black and reddish morphs of the coral trout Plectropomus leopardus. High-throughput sequencing generated a total of 958,614 sequence reads covering 164.80 Mb of two mitogenomes with a coverage of 4800X. Thirty-seven mitochondrial genes and gene order of P. leopardus was quite similar to that of other teleostean fishes. Most genes were either abutted or overlapped, and all the protein-coding genes began with an ATG start codon except for COX1 and ATP6. The number of stop codon was different for the black and reddish P. leopardus. Comparisons between the mitochondrial sequences of the two morphs revealed a total of 74 variable sites and one indel. Nucleotide diversity across protein-coding gene varied from 0.0006 (16s rRNA) to 0.0070 (Cytochrome b). As expected, the highest level of nucleotide diversity (0.0291) was detected in the control region. Our results demonstrate the NGS technology based on Ion torrent platform can be used to assemble the mitogenome of fish species.

Parallel tagged amplicon sequencing of transcriptome‐based genetic markers for Triturus newts with the Ion Torrent next‐generation sequencing platform

Next-generation sequencing is a fast and cost-effective way to obtain sequence data for nonmodel organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next, these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently, individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next, we test the utility of our markers. baps allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. newhybrids, a hybrid index and hiest confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus.

High Similarity of Novel Orthoreovirus Detected in a Child Hospitalized with Acute Gastroenteritis to Mammalian Orthoreoviruses Found in Bats in Europe

Mammalian orthoreoviruses (MRVs) are known to cause mild enteric and respiratory infections in humans. They are widespread and infect a broad spectrum of mammals. We report here the first case of an MRV detected in a child with acute gastroenteritis, which showed the highest similarity to an MRV reported recently in European bats. An examination of a stool sample from the child was negative for most common viral and bacterial pathogens. Reovirus particles were identified by electron microscopic examination of both the stool suspension and cell culture supernatant. The whole-genome sequence was obtained with the Ion Torrent next-generation sequencing platform. Prior to sequencing, the stool sample suspension and cell culture supernatant were pretreated with nucleases and/or the convective interaction medium (CIM) monolithic chromatographic method to purify and concentrate the target viral nucleic acid. Whole-genome sequence analysis revealed that the Slovenian SI-MRV01 isolate was most similar to an MRV found in a bat in Germany. High similarity was shared in all genome segments, with nucleotide and amino acid identities between 93.8 to 99.0% and 98.4 to 99.7%, respectively. It was shown that CIM monolithic chromatography alone is an efficient method for enriching the sample in viral particles before nucleic acid isolation and next-generation sequencing application.

A pragmatic approach to the analysis of diets of generalist predators: the use of next‐generation sequencing with no blocking probes

Predicting whether a predator is capable of affecting the dynamics of a prey species in the field implies the analysis of the complete diet of the predator, not simply rates of predation on a target taxon. Here, we employed the Ion Torrent next-generation sequencing technology to investigate the diet of a generalist arthropod predator. A complete dietary analysis requires the use of general primers, but these will also amplify the predator unless suppressed using a blocking probe. However, blocking probes can potentially block other species, particularly if they are phylogenetically close. Here, we aimed to demonstrate that enough prey sequence could be obtained without blocking probes. In communities with many predators, this approach obviates the need to design and test numerous blocking primers, thus making analysis of complex community food webs a viable proposition. We applied this approach to the analysis of predation by the linyphiid spider Oedothorax fuscus in an arable field. We obtained over two million raw reads. After discarding the low-quality and predator reads, the libraries still contained over 61000 prey reads (3% of the raw reads; 6% of reads passing quality control). The libraries were rich in Collembola, Lepidoptera, Diptera and Nematoda. They also contained sequences derived from several spider species and from horticultural pests (aphids). Oedothorax fuscus is common in UK cereal fields, and the results showed that it is exploiting a wide range of prey. Next-generation sequencing using general primers but without blocking probes provided ample sequences for analysis of the prey range of this spider and proved to be a simple and inexpensive approach.

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.

Isolation and characterization of 21 polymorphic microsatellite loci in the iconic Australian lungfish, Neoceratodus forsteri, using the Ion Torrent next-generation sequencing platform

We isolated and characterized 21 microsatellite loci in the vulnerable and iconic Australian lungfish, Neoceratodus forsteri. Loci were screened across eight individuals from the Burnett River and 40 individuals from the Pine River. Genetic diversity was low with between one and six alleles per locus within populations and a maximum expected heterozygosity of 0.774. These loci will now be available to assess effective population sizes and genetic structure in N. forsteri across its natural range in South East Queensland, Australia.

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.