Ancestry and phenotype predictions from touch DNA using massively parallel sequencing.

Direct PCR can be used to successfully generate full STR profiles from DNA present on the surface of objects. STR profiles are only of use in cases where a potential donor profile is available for comparison, and DNA is of sufficient DNA quality and quantity to generate a reliable profile. Often, no donor information is available and only trace DNA is present on items. As a result, alternative techniques are required to generate genetic data that can provide investigative leads. Massively parallel sequencing (MPS) offers the ability to detect trace levels of DNA and improve DNA analysis success from touched items. Here, we present the first application of direct PCR coupled with MPS to generate forensic intelligence SNP data from latent DNA. The panels assessed are (1) the HIrisplex System that targets 24 SNPs to simultaneously predict hair and eye, and (2) the Precision ID Ancestry Panel that targets 165 autosomal SNPs indicative of biogeographic ancestry. For each panel, we analysed 60 touched samples across five individuals and four substrates (glass slide, fuse, zip-lock bag and wire) using Ion AmpliSeq Library Preparation Kit on the automated Ion Chef System and Ion Torrent PGM. We examine the SNP recovery, concordance with reference samples and the genotype reproducibility from different substrates and donors. The results demonstrate the application of this approach for obtaining informative genetic from trace amounts of DNA.

Variant call concordance between two laboratory-developed, solid tumor targeted genomic profiling assays using distinct workflows and sequencing instruments.

Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard methodology in clinical laboratories for detecting somatic variants in solid tumors. The variety of methodologies and sequencing platforms in the marketplace for TGP has resulted in a variety of clinical TGP laboratory developed tests (LDT). The variability of LDTs is a challenge for test-to-test and laboratory-to-laboratory reliability. At the University of Vermont Medical Center (UVMMC), we validated a TGP assay for solid tumors which utilizes DNA hybridization capture and complete exon and selected intron sequencing of 29 clinically actionable genes. The validation samples were run on the Illumina MiSeq platform. Clinical specificity and sensitivity were evaluated by testing samples harboring genomic variants previously identified in CLIA-approved, CAP accredited laboratories with clinically validated molecular assays. The Molecular Laboratory at Dartmouth Hitchcock Medical Center (DHMC) provided 11 FFPE specimens that had been analyzed on AmpliSeq Cancer Hotspot Panel version 2 (CHPv2) and run on the Ion Torrent PGM. A Venn diagram of the gene lists from the two institutions is shown. This provided an excellent opportunity to compare the inter-laboratory reliability using two different target sequencing methods and sequencing platforms. Our data demonstrated an exceptionally high level of concordance with respect to the sensitivity and specificity of the analyses. All clinically-actionable SNV and InDel variant calls in genes covered by both panels (n=17) were identified by both laboratories. This data supports the proposal that distinct gene panel designs and sequencing workflows are capable of making consistent variant calls in solid tumor FFPE-derived samples.

Dynamics of circulating tumor DNA represented by the activating and resistant mutations in epidermal growth factor receptor tyrosine kinase inhibitor treatment.

Circulating tumor DNA (ctDNA) is an emerging field of cancer research. For lung cancer, non-invasive genotyping of EGFR is the foremost application. The activating mutations represent the ctDNA from all cancer cells, and the T790M-resistant mutation represents that from resistant cells. We examined the ctDNA dynamics of EGFR mutations by using deep sequencing with a massively parallel DNA sequencer. We obtained 190 plasma samples from 57 patients at various times during the treatment course and classified them according to treatment status. The mutation detection rate of exon 19 deletion/L858R in plasma was high at the initiation of treatment with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI; P = 0.001), suppressed during EGFR-TKI treatment before disease progression, and elevated after the onset of disease progression (P = 0.023). The mutation detection rate of T790M was low until the onset of disease progression and elevated thereafter (P = 0.01). Samples across the development of disease progression were obtained from 10 patients and showed a correlation between increased ctDNA level and disease progression. Decreased ctDNA level in response to the initiation of EGFR-TKI was observed in 4 of 6 eligible patients. In two patients, the ctDNA dynamics suggested the presence of cancer cell populations only with the T790M mutation. In another patient, the T790M ctDNA represented cell subpopulations that respond to cytotoxic agents differently from the major population. Considering the high incidence, ctDNA could be a clinical parameter to complement information from image analyses.

Exome and genome sequencing: a revolution for the discovery and diagnosis of monogenic disorders.

Massively parallel DNA sequencing has revolutionized analyses of human genetic variation. From having been out of reach for individual research groups and even more so for clinical diagnostic laboratories until recently, it is now possible to analyse complete human genomes within reasonable time frames and at a reasonable cost using technologies that are becoming increasingly available. This represents a huge advance in our ability to provide correct diagnoses for patients with rare inherited disorders and their families. This paradigm shift is especially dramatic within the area of monogenic disorders. Not only can rapid and safe diagnostics of virtually all known single-gene defects now be established, but novel causes of disease in previously unsolved cases can also be identified, illuminating novel pathways important for normal physiology. This greatly increases the capability not only to improve management of rare disorders, but also to improve understanding of pathogenetic mechanisms relevant for common, complex diseases.

Novel PTPRQ mutations identified in three congenital hearing loss patients with various types of hearing loss.

OBJECTIVES: We present 3 patients with congenital sensorineural hearing loss (SNHL) caused by novel PTPRQ mutations, including clinical manifestations and phenotypic features. METHODS: Two hundred twenty (220) Japanese subjects with SNHL from unrelated and nonconsanguineous families were enrolled in the study. Targeted genomic enrichment with massively parallel DNA sequencing of all known nonsyndromic hearing loss genes was performed to identify the genetic cause of hearing loss. RESULTS: Four novel causative PTPRQ mutations were identified in 3 cases. Case 1 had progressive profound SNHL with a homozygous nonsense mutation. Case 2 had nonprogressive profound SNHL with a compound heterozygous mutation (nonsense and missense mutation). Case 3 had nonprogressive moderate SNHL with a compound heterozygous mutation (missense and splice site mutation). Caloric test and vestibular evoked myogenic potential (VEMP) test showed vestibular dysfunction in Case 1. CONCLUSION: Hearing loss levels and progression among the present cases were varied, and there seem to be no obvious correlations between genotypes and the phenotypic features of their hearing loss. The PTPRQ mutations appeared to be responsible for vestibular dysfunction.

Deafness gene variations in a 1120 nonsyndromic hearing loss cohort: molecular epidemiology and deafness mutation spectrum of patients in Japan.

OBJECTIVES: To elucidate the molecular epidemiology of hearing loss in a large number of Japanese patients analyzed using massively parallel DNA sequencing (MPS) of target genes. METHODS: We performed MPS of target genes using the Ion PGM system with the Ion AmpliSeq and HiSeq 2000 systems using SureSelect in 1389 samples (1120 nonsyndromic hearing loss cases and 269 normal hearing controls). We filtered the variants identified using allele frequencies in a large number of controls and 12 predication program scores. RESULTS: We identified 8376 kinds of variants in the 1389 samples, and 409 835 total variants were detected. After filtering the variants, we selected 2631 kinds of candidate variants. The number of GJB2 mutations was exceptionally high among these variants, followed by those in CDH23, SLC26A4, MYO15A, COL11A2, MYO7A, and OTOF. CONCLUSIONS: We performed a large number of MPS analyses and clarified the genetic background of Japanese patients with hearing loss. This data set will be a powerful tool to discover rare causative gene mutations in highly heterogeneous monogenic diseases and reveal the genetic epidemiology of deafness.

Massively parallel DNA sequencing successfully identified seven families with deafness-associated MYO6 mutations: the mutational spectrum and clinical characteristics.

OBJECTIVES: To elucidate the involvement of MYO6 mutations, known to be responsible for DFNA22/DFNB37, in Japanese hearing loss patients through the use of genetic analysis. METHODS: Genomic variations responsible for hearing loss were identified by massively parallel DNA sequencing (MPS) of 63 target candidate genes in 1120 Japanese hearing loss patients, and the detailed clinical features for the patients with MYO6 mutations were collected and analyzed. RESULTS: Four mutations were successfully found in 7 families exhibiting autosomal dominant inheritance. All of the patients showed progressive hearing loss, but hearing type and onset age varied. Further, none of the affected patients showed any associated symptoms, such as hypertrophic cardiomyopathy or retinitis pigmentosa. CONCLUSIONS: MPS is powerful tool for the identification of rare causative deafness gene mutations, such as MYO6. The clinical characteristics noted in the present study not only confirmed the findings of previous reports but provided important new clinical information.

Mutations in the MYO15A gene are a significant cause of nonsyndromic hearing loss: massively parallel DNA sequencing-based analysis.

OBJECTIVES: Screening for MYO15A mutations was carried out using a large cohort to clarify the frequency and clinical characteristics of patients with MYO15A (DFNB3) mutations in a hearing loss population. METHODS: Genetic analysis of 63 previously reported deafness genes based on massively parallel DNA sequencing (MPS) in 1120 Japanese hearing loss patients from 53 otorhinolaryngology departments was performed. Detailed clinical features of the patients with MYO15A mutations were then collected and analyzed. RESULTS: Eleven patients from 10 families were found to have compound heterozygosity for MYO15A. Audiograms showed profound or high frequency hearing loss, with some patients showing progressive hearing loss. Age at onset was found to vary from 0 to 14 years, which seemed to be associated with the mutation. Four children underwent bilateral cochlear implantation for congenital hearing loss, with all showing good results. CONCLUSION: Mutations in the MYO15A gene are a notable cause of nonsyndromic hearing loss. MPS technology successfully detected mutations in relatively rare deafness genes such as MYO15A.

Mutational spectrum and clinical features of patients with ACTG1 mutations identified by massively parallel DNA sequencing.

OBJECTIVES: ACTG1 has been reported to be a causative gene for autosomal dominant sensorineural hearing loss, DFNA20/26. In this study we sought to clarify the detailed mutational spectrum, clinical features, and genotype-phenotype correlations. METHODS: Massively parallel DNA sequencing (MPS) of 63 target candidate genes was used to screen 1120 Japanese hearing loss patients. RESULTS: MPS screening successfully identified 4 ACTG1 mutations in 5 families. The majority of patients showed high frequency-involved progressive hearing loss, with the age of onset mostly in the first or second decade. One patient received electric acoustic stimulation (EAS), which showed a good outcome. CONCLUSIONS: Target exon-sequencing using MPS was proven to be a powerful new clinical diagnostic tool for the identification of rare causative genes such as ACTG1. The present clinical findings not only confirmed those previous reports but also provided important new clinical information.

The patients associated with TMPRSS3 mutations are good candidates for electric acoustic stimulation.

OBJECTIVES: To clarify the frequency of TMPRSS3 mutations in the hearing loss population, genetic analysis was performed, and detailed clinical characteristics were collected. Optical intervention for patients with TMPRSS3 mutations was also discussed. METHODS: Massively parallel DNA sequencing (MPS) was applied for the target exon-sequencing of 63 deafness genes in a population of 1120 Japanese hearing loss patients. RESULTS: Hearing loss in 5 patients was found to be caused by compound heterozygous TMPRSS3 mutations, and their detailed clinical features were collected and analyzed. Typically, all of the patients showed ski slope type audiograms and progressive hearing loss. Three of the 5 patients received electric acoustic stimulation (EAS), which showed good results. Further, the onset age was found to vary, and there were some correlations between genotype and phenotype (onset age). CONCLUSIONS: MPS is a powerful tool for the identification of rare causative deafness genes, such as TMPRSS3. The present clinical characteristics not only confirmed the findings from previous studies but also provided clinical evidence that EAS is beneficial for patients possessing TMPRSS3 mutations.

Functional Metagenomics to Study Antibiotic Resistance.

The construction and screening of metagenomic expression libraries have a great potential to identify novel genes with desired functions. Here, we describe metagenomic library preparation from fecal DNA, screening of libraries for antibiotic resistance genes (ARGs), massively parallel DNA sequencing of the enriched DNA fragments, and a computational pipeline for high-throughput assembly and annotation of functionally selected DNA.

Performance Comparison of Massively Parallel Sequencing (MPS) Instruments Using Single-Nucleotide Polymorphism (SNP) Panels for Ancestry.

Thermo Fisher Scientific released the Precision ID Ancestry Panel, a 165-single-nucleotide polymorphism (SNP) panel for ancestry prediction that was initially compatible with the manufacturer's massively parallel sequencer, the Ion Torrent Personal Genome Machine (PGM). The semiautomated workflow using the panel with the PGM involved several time-consuming manual steps across three instruments, including making templating solutions and loading sequencing chips. In 2014, the manufacturer released the Ion Chef robot, followed by the Ion S5 massively parallel sequencer in late 2015. The robot performs the templating with reagent cartridges and loads the chips, thus creating a fully automated workflow across two instruments. The objective of the work reported here is to compare the performance of two massively parallel sequencing systems and ascertain if the change in the workflow produces different ancestry predictions. For performance comparison of the two systems, forensic-type samples (n = 16) were used to make libraries. Libraries were templated either with the Ion OneTouch 2 system (for the PGM) or on the Ion Chef robot (for the S5). Sequencing results indicated that the ion sphere particle performance metrics were similar for the two systems. The total coverages per SNP and SNP quality were both higher for the S5 system. Ancestry predictions were concordant for the mock forensic-type samples sequenced on both massively parallel sequencing systems. The results indicated that automating the workflow with the Ion Chef system reduced the labor involved and increased the sequencing quality.

Prevalence and Clinical Characteristics of Hearing Loss Caused by MYH14 Variants.

Variants in MYH14 are reported to cause autosomal dominant nonsyndromic hereditary hearing loss (ADNSHL), with 34 variants reported to cause hearing loss in various ethnic groups. However, the available information on prevalence, as well as with regard to clinical features, remains fragmentary. In this study, genetic screening for MYH14 variants was carried out using a large series of Japanese hearing-loss patients to reveal more detailed information. Massively parallel DNA sequencing of 68 target candidate genes was applied in 8074 unrelated Japanese hearing-loss patients (including 1336 with ADNSHL) to identify genomic variations responsible for hearing loss. We identified 11 families with 10 variants. The prevalence was found to be 0.14% (11/8074) among all hearing-loss patients and 0.82% (11/1336) among ADNSHL patients. Nine of the eleven variants identified were novel. The patients typically showed late-onset hearing loss arising later than 20 years of age (64.3%, 9/14) along with progressive (92.3%, 12/13), moderate (62.5%, 10/16), and flat-type hearing loss (68.8%, 11/16). We also confirmed progressive hearing loss in serial audiograms. The clinical information revealed by the present study will contribute to further diagnosis and management of MYH14-associated hearing loss.

Development of a high efficient promoter finding method based on transient transfection.

In metazoan genome, the mechanism of gene expression regulation between transcriptional regulatory elements and their target gene is spatiotemporal. Active promoters possess many specific chromosomal features, such as hypersensitive to DNaseI and enrichment of specific histone modifications. In this article, we proposed a novel method which possesses a high efficiency to find promoters in vitro. A promoter-trap library was constructed with totally 706 random mouse genomic DNA fragment clones, and 260 promoter-active fragments of the library were screened by transient transfection into 4T1 cells. To demonstrate the accuracy of this promoter finding method, 13 fragments with promoter activities were randomly selected for published DNase-seq and ChIP-seq data analysis, downstream transcripts prediction and expression confirmation. qRT-PCR results showed that six predicted transcription units were successfully amplified in different mouse tissues/cells or in reconstituted mouse mammary tumors. Our results indicate that this promoter finding method can successfully detect the promoter-active fragments and their downstream transcripts.

MicroRNA Sequencing Data Analysis Toolkits.

MicroRNA (miRNA) is a non-protein-coding small RNA molecule that negatively regulates gene expression by degradation of mRNA or suppression of mRNA translation. MiRNAs play important roles in biological processes such as cellular development, differentiation, proliferation, apoptosis and stem cell self-renewal and cancer development. The expression profile of microRNAs is tissue-, cell-type specific. PCR- and microarray-based arrays are the commonly used for differential expression of microRNAs between different diseased conditions. With the next-generation sequencing or massively parallel DNA sequencing technology advanced and the cost is plummeting, microRNAseq approach is widely used for the genome-wide discovery of known and unknown miRNA expression. However, the analysis of microRNAseq is computational expensive, here I provide guidelines and available tools for microRNAseq analysis.

Optimization of the Promega PowerSeq™ Auto/Y system for efficient integration within a forensic DNA laboratory.

The application of massively parallel sequencing (MPS) is growing in the forensic DNA field, as forensic DNA laboratories are continuously seeking methods to gain information from a limited or degraded forensic sample. However, the laborious nature of current MPS methodologies required for successful library preparation and sequencing leave opportunities for improvement to make MPS a practical option for processing forensic casework. In this study, the Promega PowerSeq™ Auto/Y System Prototype, a MPS laboratory workflow that incorporates multiplex amplification, was selected for optimization with the objectives to introduce automation for relieving manual processing, and to reduce the number of steps recommended by the standard protocol. Successful changes in the optimized workflow included a switch from column-based PCR purification to automatable bead-based purification, adoption of the library preparation procedures by a liquid handling robot platform, and removal of various time-consuming quality checks. All data in this study were found to be concordant with capillary electrophoresis (CE) data and previously-generated MPS results from this workflow. Read abundance and allele balance, metrics related to sample interpretation reliability, were not significantly different when compared to samples processed with the manufacturer's protocol. All the modifications implemented resulted in increased laboratory efficiency, reduced the protocol steps associated with risk of contamination and human error events, and decreased manual processing time by approximately 12h. These findings provide forensic DNA laboratories a more streamlined option when considering implementation of a MPS workflow.

Functional Metagenomics to Study Antibiotic Resistance.

The construction and screening of metagenomic expression libraries has great potential to identify novel genes and their functions. Here, we describe metagenomic library preparation from fecal DNA, screening of libraries for antibiotic resistance genes (ARGs), massively parallel DNA sequencing of the enriched DNA fragments, and a computational pipeline for high-throughput assembly and annotation of functionally selected DNA.

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA.

Second-generation sequencing of forensic STRs using the Ion Torrent™ HID STR 10-plex and the Ion PGM™.

Second-generation sequencing (SGS) using Roche/454 and Illumina platforms has proved capable of sequencing the majority of the key forensic genetic STR systems. Given that Roche has announced that the 454 platforms will no longer be supported from 2015, focus should now be shifted to competing SGS platforms, such as the MiSeq (Illumina) and the Ion Personal Genome Machine (Ion PGM™; Thermo Fisher). There are currently several challenges faced with amplicon-based SGS STR typing in forensic genetics, including current lengths of amplicons for CE-typing and lack of uniform data analysis between laboratories. Thermo Fisher has designed a human identification (HID) short tandem repeat (STR) 10-plex panel including amelogenin, CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX and vWA, where the primers have been designed specifically for the purpose of SGS and the data analysis is supported by Ion Torrent™ software. Hence, the combination of the STR 10-plex and the Ion PGM™ represents the first fully integrated SGS STR typing solution from PCR to data analysis. In this study, four experiments were performed to evaluate the alpha-version of the STR 10-plex: (1) typing of control samples; (2) analysis of sensitivity; (3) typing of mixtures; and (4) typing of biological crime case samples. Full profiles and concordant results between replicate SGS runs and CE-typing were observed for all control samples. Full profiles were seen with DNA input down to 50 pg, with the exception of a single locus drop-out in one of the 100 pg dilutions. Mixtures were easily deconvoluted down to 20:1, although alleles from the minor contributor had to be identified manually as some signals were not called by the Ion Torrent™ software. Interestingly, full profiles were obtained for all biological samples from real crime and identification cases, in which only partial profiles were obtained with PCR-CE assays. In conclusion, the Ion Torrent™ HID STR 10-plex panel offers an all-in-one solution from amplification of STRs and amelogenin, and sequencing to data analysis.

Sequencing of the Dutch elm disease fungus genome using the Roche/454 GS-FLX Titanium System in a comparison of multiple genomics core facilities.

As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30-50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs.