Identification of EMS-Induced Mutations in Drosophila melanogaster by Whole-Genome Sequencing

Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing ~70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods.     

基于高通量测序技术的微生物检测数据分析方法

高通量测序技术的发展正在逐渐改变诸多生物学领域的研究方法.为应对突发疫情以及新发未知微生物威胁的需求,微生物鉴定技术逐渐从传统的物理化学方法及核酸杂交等分子水平方法进一步走向利用无需培养的测序数据进行快速分析检测.随之而来的是对高通量数据分析在精度及速度的要求.基于高通量测序数据的微生物检测数据分析方法在近些年得到了快速的发展.本文分析了目前基于高通量测序数据的微生物检测数据分析方法,对其数据分析的处理流程和计算方法进行了研究,比较了各个微生物检测数据分析方法的特点及适用场景.最后结合本实验室工作总结微生物检测数据分析方法在实际应用中可能遇到的问题,希望对该应用领域的研究有一定的参考意义.     

Efficient Detection of Mutations in Wilson Disease by Manifold Sequencing

We have applied a solid support for parallel handling and direct loading of sequencing reactions—manifold sequencing—to analyze the coding sequence for the deficient copper transporting P-type ATPase in 24 families with Wilson disease. At least 100 different amplification reactions could be handled in parallel, with a minimal turnaround time of 12 h from isolated genomic DNA to identification of the mutations. Sixteen different mutations were found, accounting for 92% of the mutant genes. Ten of these mutations have not been previously described. Eleven were observed only in single families. Mutation His1069Gln, previously identified as the most prevalent mutation in Northern Europe, was found in one-third of the Northern European chromosomes in our material. Four patients were homozygous for this mutation, and three were homozygous for Thr977Met. The method allowed us to establish the diagnosis of Wilson disease in 24 h in a patient with acute hepatic failure.     

Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.     

Development and Evaluation of a Panel of Filovirus Sequence Capture Probes for Pathogen Detection by Next-Generation Sequencing

A detailed understanding of the circulating pathogens in a particular geographic location aids in effectively utilizing targeted, rapid diagnostic assays, thus allowing for appropriate therapeutic and containment procedures. This is especially important in regions prevalent for highly pathogenic viruses co-circulating with other endemic pathogens such as the malaria parasite. The importance of biosurveillance is highlighted by the ongoing Ebola virus disease outbreak in West Africa. For example, a more comprehensive assessment of the regional pathogens could have identified the risk of a filovirus disease outbreak earlier and led to an improved diagnostic and response capacity in the region. In this context, being able to rapidly screen a single sample for multiple pathogens in a single tube reaction could improve both diagnostics as well as pathogen surveillance. Here, probes were designed to capture identifying filovirus sequence for the ebolaviruses Sudan, Ebola, Reston, Taï Forest, and Bundibugyo and the Marburg virus variants Musoke, Ci67, and Angola. These probes were combined into a single probe panel, and the captured filovirus sequence was successfully identified using the MiSeq next-generation sequencing platform. This panel was then used to identify the specific filovirus from nonhuman primates experimentally infected with Ebola virus as well as Bundibugyo virus in human sera samples from the Democratic Republic of the Congo, thus demonstrating the utility for pathogen detection using clinical samples. While not as sensitive and rapid as real-time PCR, this panel, along with incorporating additional sequence capture probe panels, could be used for broad pathogen screening and biosurveillance.     

Exome Sequencing Identifies INPPL1 Mutations as a Cause of Opsismodysplasia

Opsismodysplasia (OPS) is a severe autosomal-recessive chondrodysplasia characterized by pre- and postnatal micromelia with extremely short hands and feet. The main radiological features are severe platyspondyly, squared metacarpals, delayed skeletal ossification, and metaphyseal cupping. In order to identify mutations causing OPS, a total of 16 cases (7 terminated pregnancies and 9 postnatal cases) from 10 unrelated families were included in this study. We performed exome sequencing in three cases from three unrelated families and only one gene was found to harbor mutations in all three cases: inositol polyphosphate phosphatase-like 1 (INPPL1). Screening INPPL1 in the remaining cases identified a total of 12 distinct INPPL1 mutations in the 10 families, present at the homozygote state in 7 consanguinous families and at the compound heterozygote state in the 3 remaining families. Most mutations (6/12) resulted in premature stop codons, 2/12 were splice site, and 4/12 were missense mutations located in the catalytic domain, 5-phosphatase. INPPL1 belongs to the inositol-1,4,5-trisphosphate 5-phosphatase family, a family of signal-modulating enzymes that govern a plethora of cellular functions by regulating the levels of specific phosphoinositides. Our finding of INPPL1 mutations in OPS, a severe spondylodysplastic dysplasia with major growth plate disorganization, supports a key and specific role of this enzyme in endochondral ossification.     

Efficient detection of viral transmissions with Next-Generation Sequencing data

Background

Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections associated with unsafe injection practices, drug diversion, and other exposures to blood are difficult to detect and investigate. Molecular analysis has been frequently used in the study of HCV outbreaks and transmission chains; helping identify a cluster of sequences as linked by transmission if their genetic distances are below a previously defined threshold. However, HCV exists as a population of numerous variants in each infected individual and it has been observed that minority variants in the source are often the ones responsible for transmission, a situation that precludes the use of a single sequence per individual because many such transmissions would be missed.The use of Next-Generation Sequencing immensely increases the sensitivity of transmission detection but brings a considerable computational challenge because all sequences need to be compared among all pairs of samples.

Methods

We developed a three-step strategy that filters pairs of samples according to different criteria: (i) a k-mer bloom filter, (ii) a Levenhstein filter and (iii) a filter of identical sequences. We applied these three filters on a set of samples that cover the spectrum of genetic relationships among HCV cases, from being part of the same transmission cluster, to belonging to different subtypes.

Results

Our three-step filtering strategy rapidly removes 85.1% of all the pairwise sample comparisons and 91.0% of all pairwise sequence comparisons, accurately establishing which pairs of HCV samples are below the relatedness threshold.

Conclusions

We present a fast and efficient three-step filtering strategy that removes most sequence comparisons and accurately establishes transmission links of any threshold-based method. This highly efficient workflow will allow a faster response and molecular detection capacity, improving the rate of detection of viral transmissions with molecular data.

     

Phylogenetic Properties of 50 Nuclear Loci in Medicago (Leguminosae) Generated Using Multiplexed Sequence Capture and Next-Generation Sequencing

Next-generation sequencing technology has increased the capacity to generate molecular data for plant biological research, including phylogenetics, and can potentially contribute to resolving complex phylogenetic problems. The evolutionary history of Medicago L. (Leguminosae: Trifoliae) remains unresolved due to incongruence between published phylogenies. Identification of the processes causing this genealogical incongruence is essential for the inference of a correct species phylogeny of the genus and requires that more molecular data, preferably from low-copy nuclear genes, are obtained across different species. Here we report the development of 50 novel LCN markers in Medicago and assess the phylogenetic properties of each marker. We used the genomic resources available for Medicago truncatula Gaertn., hybridisation-based gene enrichment (sequence capture) techniques and Next-Generation Sequencing to generate sequences. This alternative proves to be a cost-effective approach to amplicon sequencing in phylogenetic studies at the genus or tribe level and allows for an increase in number and size of targeted loci. Substitution rate estimates for each of the 50 loci are provided, and an overview of the variation in substitution rates among a large number of low-copy nuclear genes in plants is presented for the first time. Aligned sequences of major species lineages of Medicago and its sister genus are made available and can be used in further probe development for sequence-capture of the same markers.     

Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing

Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10^-5). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.     

High-Throughput Discovery of Mutations in Tef Semi-Dwarfing Genes by Next-Generation Sequencing Analysis

Tef (Eragrostis tef) is a major cereal crop in Ethiopia. Lodging is the primary constraint to increasing productivity in this allotetraploid species, accounting for losses of ∼15–45% in yield each year. As a first step toward identifying semi-dwarf varieties that might have improved lodging resistance, an ∼6× fosmid library was constructed and used to identify both homeologues of the dw3 semi-dwarfing gene of Sorghum bicolor. An EMS mutagenized population, consisting of ∼21,210 tef plants, was planted and leaf materials were collected into 23 superpools. Two dwarfing candidate genes, homeologues of dw3 of sorghum and rht1 of wheat, were sequenced directly from each superpool with 454 technology, and 120 candidate mutations were identified. Out of 10 candidates tested, six independent mutations were validated by Sanger sequencing, including two predicted detrimental mutations in both dw3 homeologues with a potential to improve lodging resistance in tef through further breeding. This study demonstrates that high-throughput sequencing can identify potentially valuable mutations in under-studied plant species like tef and has provided mutant lines that can now be combined and tested in breeding programs for improved lodging resistance.     

Exome Sequencing Reveals Novel and Recurrent Mutations with Clinical Significance in Inherited Retinal Dystrophies

This study aimed to identify the underlying molecular genetic cause in four Spanish families clinically diagnosed of Retinitis Pigmentosa (RP), comprising one autosomal dominant RP (adRP), two autosomal recessive RP (arRP) and one with two possible modes of inheritance: arRP or X-Linked RP (XLRP). We performed whole exome sequencing (WES) using NimbleGen SeqCap EZ Exome V3 sample preparation kit and SOLID 5500xl platform. All variants passing filter criteria were validated by Sanger sequencing to confirm familial segregation and the absence in local control population. This strategy allowed the detection of: (i) one novel heterozygous splice-site deletion in RHO, c.937-2_944del, (ii) one rare homozygous mutation in C2orf71, c.1795T>C; p.Cys599Arg, not previously associated with the disease, (iii) two heterozygous null mutations in ABCA4, c.2041C>T; p.R681* and c.6088C>T; p.R2030*, and (iv) one mutation, c.2405-2406delAG; p.Glu802Glyfs*31 in the ORF15 of RPGR. The molecular findings for RHO and C2orf71 confirmed the initial diagnosis of adRP and arRP, respectively, while patients with the two ABCA4 mutations, both previously associated with Stargardt disease, presented symptoms of RP with early macular involvement. Finally, the X-Linked inheritance was confirmed for the family with the RPGR mutation. This latter finding allowed the inclusion of carrier sisters in our preimplantational genetic diagnosis program.     

The Accuracy,Feasibility and Challenges of Sequencing Short Tandem Repeats Using Next-Generation Sequencing Platforms

To date we have little knowledge of how accurate next-generation sequencing (NGS) technologies are in sequencing repetitive sequences beyond known limitations to accurately sequence homopolymers. Only a handful of previous reports have evaluated the potential of NGS for sequencing short tandem repeats (microsatellites) and no empirical study has compared and evaluated the performance of more than one NGS platform with the same dataset. Here we examined yeast microsatellite variants from both long-read (454-sequencing) and short-read (Illumina) NGS platforms and compared these to data derived through Sanger sequencing. In addition, we investigated any locus-specific biases and differences that might have resulted from variability in microsatellite repeat number, repeat motif or type of mutation. Out of 112 insertion/deletion variants identified among 45 microsatellite amplicons in our study, we found 87.5% agreement between the 454-platform and Sanger sequencing in frequency of variant detection after Benjamini-Hochberg correction for multiple tests. For a subset of 21 microsatellite amplicons derived from Illumina sequencing, the results of short-read platform were highly consistent with the other two platforms, with 100% agreement with 454-sequencing and 93.6% agreement with the Sanger method after Benjamini-Hochberg correction. We found that the microsatellite attributes copy number, repeat motif and type of mutation did not have a significant effect on differences seen between the sequencing platforms. We show that both long-read and short-read NGS platforms can be used to sequence short tandem repeats accurately, which makes it feasible to consider the use of these platforms in high-throughput genotyping. It appears the major requirement for achieving both high accuracy and rare variant detection in microsatellite genotyping is sufficient read depth coverage. This might be a challenge because each platform generates a consistent pattern of non-uniform sequence coverage, which, as our study suggests, may affect some types of tandem repeats more than others.     

XLID-Causing Mutations and Associated Genes Challenged in Light of Data From Large-Scale Human Exome Sequencing

Because of the unbalanced sex ratio (1.3–1.4 to 1) observed in intellectual disability (ID) and the identification of large ID-affected families showing X-linked segregation, much attention has been focused on the genetics of X-linked ID (XLID). Mutations causing monogenic XLID have now been reported in over 100 genes, most of which are commonly included in XLID diagnostic gene panels. Nonetheless, the boundary between true mutations and rare non-disease-causing variants often remains elusive. The sequencing of a large number of control X chromosomes, required for avoiding false-positive results, was not systematically possible in the past. Such information is now available thanks to large-scale sequencing projects such as the National Heart, Lung, and Blood (NHLBI) Exome Sequencing Project, which provides variation information on 10,563 X chromosomes from the general population. We used this NHLBI cohort to systematically reassess the implication of 106 genes proposed to be involved in monogenic forms of XLID. We particularly question the implication in XLID of ten of them (AGTR2, MAGT1, ZNF674, SRPX2, ATP6AP2, ARHGEF6, NXF5, ZCCHC12, ZNF41, and ZNF81), in which truncating variants or previously published mutations are observed at a relatively high frequency within this cohort. We also highlight 15 other genes (CCDC22, CLIC2, CNKSR2, FRMPD4, HCFC1, IGBP1, KIAA2022, KLF8, MAOA, NAA10, NLGN3, RPL10, SHROOM4, ZDHHC15, and ZNF261) for which replication studies are warranted. We propose that similar reassessment of reported mutations (and genes) with the use of data from large-scale human exome sequencing would be relevant for a wide range of other genetic diseases.     

Exome Sequencing Identifies Mutations in CCDC114 as a Cause of Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ∼60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.     

Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design

BackgroundThe soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays.Conclusions/SignificanceThe utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.     

Exome Sequencing Identifies GNB4 Mutations as a Cause of Dominant Intermediate Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of inherited neuropathies. Mutations in approximately 45 genes have been identified as being associated with CMT. Nevertheless, the genetic etiologies of at least 30% of CMTs have yet to be elucidated. Using a genome-wide linkage study, we previously mapped a dominant intermediate CMT to chromosomal region 3q28–q29. Subsequent exome sequencing of two affected first cousins revealed heterozygous mutation c.158G>A (p.Gly53Asp) in GNB4, encoding guanine-nucleotide-binding protein subunit beta-4 (Gβ₄), to cosegregate with the CMT phenotype in the family. Further analysis of GNB4 in an additional 88 unrelated CMT individuals uncovered another de novo mutation, c.265A>G (p.Lys89Glu), in this gene in one individual. Immunohistochemistry studies revealed that Gβ₄ was abundant in the axons and Schwann cells of peripheral nerves and that expression of Gβ₄ was significantly reduced in the sural nerve of the two individuals carrying the c.158G>A (p.Gly53Asp) mutation. In vitro studies demonstrated that both the p.Gly53Asp and p.Lys89Glu altered proteins impaired bradykinin-induced G-protein-coupled-receptor (GPCR) signaling, which was facilitated by the wild-type Gβ₄. This study identifies GNB4 mutations as a cause of CMT and highlights the importance of Gβ₄-related GPCR signaling in peripheral-nerve function in humans.     

Identification of Parasitic Communities within European Ticks Using Next-Generation Sequencing

Background

Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents.

Methodology/Principal Findings

We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species.

Conclusions/Significance

Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.