Personal Genome Machine Sequencing Research Articles

Pulmonary Langerhans Cell Histiocytosis: Profiling of Multifocal Tumors Using Next-Generation Sequencing Identifies Concordant Occurrence of BRAF V600E Mutations

BackgroundPulmonary Langerhans cell histiocytosis is a localized proliferation of Langerhans cells in the lung that presents without systemic manifestations as bilateral nodular lung disease in adult cigarette smokers. The molecular basis for this proliferation is unknown. MethodsTwenty-two concurrent nodules in five patients were microdissected from formalin-fixed paraffin-embedded tissue and analyzed by next-generation sequencing for mutations in 46 cancer genes with the Ion AmpliSeq Cancer Panel on an Ion PGM (Personal Genome Machine) Sequencer (Life Technologies Corporation). Mutation confirmation was performed by conventional Sanger sequencing or by sensitive coamplification at lower denaturation polymerase chain reaction/fluorescence melting curve analysis. ResultsSmall amounts of DNA (10 ng) isolated from nodules were sufficient for successful interrogation of 740 mutational hot spots in 46 cancer genes by the Ion PGM Sequencer, with an average depth of coverage of 2,783 reads per hot spot and with uniformity of coverage of 92%. BRAF V600E mutation was detected in all concurrent nodules studied in two of the five patients, whereas in three of the five patients, no oncogene mutations were found. ConclusionsPulmonary Langerhans cell histiocytosis appears to be a clonal proliferation that may or may not have BRAF V600E mutations. For those with BRAF V600E mutations, new targeted therapies, such as vemurafenib, may be used in progressive cases.

Feasibility Study of Semiconductor Sequencing for Noninvasive Prenatal Detection of Fetal Aneuploidy

Noninvasive prenatal detection of common fetal aneuploidies with cell-free DNA from maternal plasma has been achieved with high-throughput next-generation sequencing platforms. Turnaround times for previously tested platforms are still unsatisfactory for clinical applications, however, because of the time spent on sequencing. The development of semiconductor sequencing technology has provided a way to shorten overall run times. We studied the feasibility of using semiconductor sequencing technology for the noninvasive detection of fetal aneuploidy. Maternal plasma DNA from 13 pregnant women, corresponding to 4 euploid, 6 trisomy 21 (T21), 2 trisomy 18 (T18), and 1 trisomy 13 (T13) pregnancies, were sequenced on the Ion Torrent Personal Genome Machine sequencer platform with 318 chips. The data were analyzed with the T statistic method after correcting for GC bias, and the T value was calculated as an indicator of fetal aneuploidy. We obtained a mean of 3 524 401 high-quality reads per sample, with an efficiency rate of 77.9%. All of the T21, T13, and T18 fetuses could be clearly distinguished from euploid fetuses, and the time spent on library preparation and sequencing was 24 h. Semiconductor sequencing represents a suitable technology for the noninvasive prenatal detection of fetal aneuploidy. With this platform, sequencing times can be substantially reduced; however, a further larger-scale study is needed to determine the imprecision of noninvasive fetal aneuploidy detection with this system.

Fast Isogenic Mapping-by-Sequencing of Ethyl Methanesulfonate-Induced Mutant Bulks

Mapping-by-sequencing (or SHOREmapping) has revitalized the powerful concept of forward genetic screens in plants. However, as in conventional genetic mapping approaches, mapping-by-sequencing requires phenotyping of mapping populations established from crosses between two diverged accessions. In addition to the segregation of the focal phenotype, this introduces natural phenotypic variation, which can interfere with the recognition of quantitative phenotypes. Here, we demonstrate how mapping-by-sequencing and candidate gene identification can be performed within the same genetic background using only mutagen-induced changes as segregating markers. Using a previously unknown suppressor of mutants of like heterochromatin protein1 (lhp1), which in its functional form is involved in chromatin-mediated gene repression, we identified three closely linked ethyl methanesulfonate-induced changes as putative candidates. In order to assess allele frequency differences between such closely linked mutations, we introduced deep candidate resequencing using the new Ion Torrent Personal Genome Machine sequencing platform to our mutant identification pipeline and thereby reduced the number of causal candidate mutations to only one. Genetic analysis of two independent additional alleles confirmed that this mutation was causal for the suppression of lhp1.

Rapid Detection of the ACMG/ACOG-Recommended 23 CFTR Disease-Causing Mutations Using Ion Torrent Semiconductor Sequencing

Cystic fibrosis (CF) is one of the most frequently diagnosed autosomal-recessive diseases in the Caucasian population. For general-population CF carrier screening, the American College of Medical Genetics (ACMG)/American College of Obstetricians and Gynecologists (ACOG) have recommended a core panel of 23 mutations that will identify 49-98% of carriers, depending on ethnic background. Using a genotyping technology that can rapidly identify disease-causing mutations is important for high-throughput general-population carrier screening, confirming clinical diagnosis, determining treatment options, and prenatal diagnosis. Here, we describe a proof-of-concept study to determine whether the Ion Torrent Personal Genome Machine (PGM) sequencer platform can reliably identify all ACMG/ACOG 23 CF transmembrane conductance regulator (CFTR) mutations. A WT CF specimen along with mutant DNA specimens representing all 23 CFTR mutations were sequenced bidirectionally on the Ion Torrent 314 chip to determine the accuracy of the PGM for CFTR variant detection. We were able to reliably identify all of the targeted mutations except for 2184delA, which lies in a difficult, 7-mer homopolymer tract. Based on our study, we believe PGM sequencing may be a suitable technology for identifying CFTR mutations in the future. However, as a result of the elevated rate of base-calling errors within homopolymer stretches, mutations within such regions currently need to be evaluated carefully using an alternative method.