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The low number and activity of hydrocarbon-degrading bacteria and the low solubility and availability of hydrocarbons hamper bioremediation of oil-contaminated soils in arid deserts, thus bioremediation treatments that circumvent these limitations are required. We tested the effect of Ethylenediaminetetraacetic acid (EDTA) addition, at different concentrations (i.e. 0.1, 1 and 10 mM), on bacterial respiration and biodegradation of Arabian light oil in bioaugmented (i.e. with the addition of exogenous alkane-degrading consortium) and non-bioaugmented oil-contaminated desert soils. Post-treatment shifts in the soils' bacterial community structure were monitored using MiSeq sequencing. Bacterial respiration, indicated by the amount of evolved CO 2 , was highest at 10 mM EDTA in bioaugmented and non-bioaugmented soils, reaching an amount of 2.2 ± 0.08 and 1.6 ± 0.02 mg-CO 2 g −1 after 14 days of incubation, respectively. GC–MS revealed that 91.5% of the C 14 –C 30 alkanes were degraded after 42 days when 10 mM EDTA and the bacterial consortium were added together. MiSeq sequencing showed that 78–91% of retrieved sequences in the original soil belonged to Deinococci , Alphaproteobacteria , Gammaproteobacteia and Bacilli . The same bacterial classes were detected in the 10 mM EDTA-treated soils, however with slight differences in their relative abundances. In the bioaugmented soils, only Alcanivorax sp. MH3 and Parvibaculum sp. MH21 from the exogenous bacterial consortium could survive until the end of the experiment. We conclude that the addition of EDTA at appropriate concentrations could facilitate biodegradation processes by increasing hydrocarbon availability to microbes. The addition of exogenous oil-degrading bacteria along with EDTA could serve as an ideal solution for the decontamination of oil-contaminated desert soils. [ABSTRACT FROM AUTHOR]

This study presents performance specifications of an in-house developed human leukocyte antigen (HLA) typing assay using next-generation sequencing (NGS) on the Illumina MiSeq platform. A total of 253 samples, previously characterized for HLA-A, -B, -C, -DRB1 and -DQB1 were included in this study, which were typed at high-resolution using a combination of Sanger sequencing, sequence-specific primer (SSP) and sequence-specific oligonucleotide probe (SSOP) technologies and recorded at the two-field level. Samples were selected with alleles that cover a high percentage of HLA specificities in each of five different race/ethnic groups: European, African-American, Asian Pacific Islander, Hispanic and Native American. Sequencing data were analyzed by two software programs, Omixon's TARGET and GenDx's NGSENGINE. A number of metrics including allele balance, sensitivity, specificity, precision, accuracy and remaining ambiguity were assessed. Data analyzed by the two software systems are shown independently. The majority of alleles were identical in the exonic sequences (third field) with both programs for HLA-A, -B, -C and -DQB1 in 97.7% of allele determinations. Among the remaining discrepant genotype calls at least one of the analysis programs agreed with the reference typing. Upon additional manual analysis 100% of the 2530 alleles were concordant with the reference HLA genotypes; the remaining ambiguities did not exceed 0.8%. The results demonstrate the feasibility and significant benefit of HLA typing by NGS as this technology is highly accurate, eliminates virtually all ambiguities, provides complete sequencing information for the length of the HLA gene and forms the basis for utilizing a single methodology for HLA typing in the immunogenetics labs. [ABSTRACT FROM AUTHOR]

Photosynthetic picoeukaryotes (PPEs) are major oceanic primary producers. However, the diversity of such communities remains poorly understood, especially in the northwestern (NW) Pacific. We investigated the abundance and diversity of PPEs, and recorded environmental variables, along a transect from the coast to the open Pacific Ocean. High-throughput tag sequencing (using the MiSeq system) revealed the diversity of plastid 16S rRNA genes. The dominant PPEs changed at the class level along the transect. Prymnesiophyceae were the only dominant PPEs in the warm pool of the NW Pacific, but Mamiellophyceae dominated in coastal waters of the East China Sea. Phylogenetically, most Prymnesiophyceae sequences could not be resolved at lower taxonomic levels because no close relatives have been cultured. Within the Mamiellophyceae, the genera Micromonas and Ostreococcus dominated in marginal coastal areas affected by open water, whereas Bathycoccus dominated in the lower euphotic depths of oligotrophic open waters. Cryptophyceae and Phaeocystis (of the Prymnesiophyceae) dominated in areas affected principally by coastal water. We also defined the biogeographical distributions of Chrysophyceae, prasinophytes, Bacillariophyceaea and Pelagophyceae. These distributions were influenced by temperature, salinity and chlorophyll a and nutrient concentrations. [ABSTRACT FROM AUTHOR]

Next-generation sequencing (NGS) has the capacity to screen a single DNA sample and detect pathogen DNA from thousands of host DNA sequence reads, making it a versatile and informative tool for investigation of pathogens in diseased animals. The technique is effective and labor saving in the initial identification of pathogens, and will complement conventional diagnostic tests to associate the candidate pathogen with a disease process. In this report, we investigated the utility of the diversity profiling NGS approach using Illumina small subunit ribosomal RNA (18S rRNA) gene amplicon deep sequencing to detect Toxoplasma gondii in previously confirmed cases of toxoplasmosis. We then tested the diagnostic approach with species-specific PCR genotyping, histopathology and immunohistochemistry of toxoplasmosis in a Risso's dolphin ( Grampus griseus ) to systematically characterise the disease and associate causality. We show that the Euk7A/Euk570R primer set targeting the V1–V3 hypervariable region of the 18S rRNA gene can be used as a species-specific assay for cyst-forming coccidia and discriminate T. gondii . Overall, the approach is cost-effective and improves diagnostic decision support by narrowing the differential diagnosis list with more certainty than was previously possible. Furthermore, it supplements the limitations of cryptic protozoan morphology and surpasses the need for species-specific PCR primer combinations. [ABSTRACT FROM AUTHOR]

Objectives In recent years, next-generation sequencing (NGS) technologies, which enable high throughput sample processing at relatively lower costs, are adopted in both research and clinical settings. A multiplex PCR-based NGS assay to identify mutations in the ATP7B gene for routine molecular diagnosis of Wilson disease was evaluated in comparison with the gold standard direct Sanger sequencing. Design and methods Five multiplex PCRs to amplify the partial promoter, 5′ untranslated and the entire coding regions of the ATP7B gene were designed. Indexed paired-end libraries were generated from the pooled amplicons using Nextera XT DNA Sample Preparation Kit and subjected to NGS on the MiSeq platform. DNA from the peripheral blood of 12 patients with Wilson disease, 2 B-lymphocyte cell lines and 3 external quality assurance samples were sequenced by the MiSeq and Sanger sequencing. Results Complete coverage was achieved across the targeted bases without any drop-out sequences. The observed read depth in a single run with 20 samples was > 100X. Comparison of the NGS results against Sanger sequencing data on a panel of clinical specimens, cell lines and European Molecular Genetics Quality Networks (EMQN) quality assurance samples showed 100% concordance in identifying pathogenic mutations. Conclusion With the capability of generating relatively higher throughput in a short time period, the NGS assay is a viable alternative to Sanger sequencing for detecting ATP7B mutations causally linked to Wilson disease in the clinical diagnostic laboratory. [ABSTRACT FROM AUTHOR]

The use of organic wastes in bioremediation of oil-contaminated desert soils has received little attention, although their use is cost-effective. We evaluated the use of spent mushroom compost (SMC), poultry manure (PM), and urea in the stimulation of respiration activities and oil degradation in a polluted desert soil. Moreover, we followed post treatment shifts in bacterial community structure using MiSeq sequencing. The addition of SMC and PM resulted in a significant increase in the evolved CO from 8.7 ± 1.9 to 25.7 ± 1.6 and to 23.4 ± 1.2 mg CO g soil after 96 days of incubation, respectively. In contrast, changes in respiration activities after the addition of urea were insignificant. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that most of the alkanes (C-C) were degraded in all biostimulated soils at a rate of 0.12-0.19 mg g soil day, which was significantly higher than in the untreated soil ( P < 0.05). Bacterial community analysis showed that 87-94 % of total sequences in the original soil belonged to Firmicutes, Actinobacteria, and Proteobacteria. While the relative abundance of Firmicutes remained unchanged after the addition of PM (37-48 % of total sequences), it increased in the urea treatment (44-87 %) and dramatically decreased in the SMC treatment (0.5-4.5 %). The remaining bacterial groups were still detectable after the treatments, although no clear treatment-related shifts could be observed, due to the large difference in the relative abundance of the same bacterial groups among the same replicates. We conclude that the use of organic wastes could be one of the ways of combating petroleum pollution in desert soils. [ABSTRACT FROM AUTHOR]

Abstract Microbial alpha-diversity, a fundamental ecological concept, is often determined using multiplexed amplicon sequencing approaches. Here we show, using standardized controls and 1879 soil samples, across 21 illumina MiSEQ high throughput sequencing (HTS) runs, that sample alpha-diversity estimates are highly dependent upon the overall diversity of all samples within the sequencing run, a likely result of within sequencing run cross-talk. Cross-talk, therefore, has grave implications for alpha-diversity interpretation in mulitplexed sequencing studies. Highlights • Microbial alpha-diversity, a fundamental ecological concept, is often determined using multiplexed amplicon sequencing approaches. • Alpha-diversity is highly dependent upon the overall diversity of all samples within the sequencing run. [ABSTRACT FROM AUTHOR]

A complete mitochondrial genome sequence was determined for a member of the Caridina indistincta species complex known as C. indistincta ‘sp. A’. The 15,461 bp sequence (GenBank: MH189850) was obtained via genome skimming, and contains 13 protein coding genes, 22 tRNA genes, 2 rRNA genes, and a 646 bp control region arranged in the pancrustacean ground pattern. Caridina indistincta sp. A is a freshwater macroinvertebrate important for ecosystem health monitoring in Australia and this reference will be a useful resource for metabarcoding and eDNA studies. [ABSTRACT FROM AUTHOR]

Introduction: Massively parallel sequencing of mitogenomes usually requires prior amplification. The PCR step may influence the quality of the data obtained, especially when low-level heteroplasmy detection is applied., Aim: The aim of this study was to compare the reliability of two different DNA polymerases in detecting homoplasmic and heteroplasmic substitutions in human mitogenomes., Material and Methods: Mitogenomes of five samples were amplified with Long PCR Enzyme Mix from Fermentas or TaKaRa LA Taq DNA Polymerase from TaKaRa. Then, NexteraTM XT DNA libraries were sequenced on MiSeq FGx platform (Illumina). mtDNA substitutions were called for alternative variants above the 1% level., Results: All homoplasmic substitutions detected in amplicons generated with polymerases studied here and sequenced on MiSeq FGx system were consistently identified as homoplasmies with alternative sequencing methods. TaKaRa LA Taq DNA Polymerase was found to be less accurate in low-level heteroplasmy detection than Long PCR Enzyme Mix enzyme as more false negative and false positive results were observed for minority variants called above the 1% level. Nevertheless, both PCR systems studied can be successfully used to detect authentic mtDNA substitutions, for which minority variants exceed the 3.61% level assuming at least 10,000x coverage and sequencing Nextera XT DNA libraries on MiSeq FGx machine., Conclusions: The accuracy and sensitivity of point heteroplasmy detection with the MiSeq FGx instrument varies on polymerase used for mtDNA amplification. Therefore, it is recommended to validate the laboratory protocols used for mtDNA substitution detection prior to their implementation for the forensic or medical genetics purposes., Competing Interests: The authors report no conflicts of interest in this work., (Copyright © 2024 by PTMSiK.)

This communication describes our experience in large-scale G group-level high resolution HLA typing using three different DNA sequencing platforms – ABI 3730 xl, Illumina MiSeq and PacBio RS II. Recent advances in DNA sequencing technologies, so-called next generation sequencing (NGS), have brought breakthroughs in deciphering the genetic information in all living species at a large scale and at an affordable level. The NGS DNA indexing system allows sequencing multiple genes for large number of individuals in a single run. Our laboratory has adopted and used these technologies for HLA molecular testing services. We found that each sequencing technology has its own strengths and weaknesses, and their sequencing performances complement each other. HLA genes are highly complex and genotyping them is quite challenging. Using these three sequencing platforms, we were able to meet all requirements for G group-level high resolution and high volume HLA typing. [ABSTRACT FROM AUTHOR]

Background: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. Results: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. Conclusions: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding. [ABSTRACT FROM AUTHOR]

Capillary electrophoresis (CE) and multiplex amplification with fluorescent tagging have been routinely used for STR typing in forensic genetics. However, CE-based methods restrict the number of markers that can be multiplexed simultaneously and cannot detect any intra-repeat variations within STRs. Several studies already have indicated that massively parallel sequencing (MPS) may be another potential technology for STR typing. In this study, the prototype PowerSeq™ Auto System (Promega) containing the 23 STR loci and amelogenin was evaluated using Illumina MiSeq. Results showed that single source complete profiles could be obtained using as little as 62 pg of input DNA. The reproducibility study showed that the profiles generated were consistent among multiple typing experiments for a given individual. The mixture study indicated that partial STR profiles of the minor contributor could be detected up to 19:1 mixture. The mock forensic casework study showed that full or partial profiles could be obtained from different types of single source and mixture samples. These studies indicate that the PowerSeq Auto System and the Illumina MiSeq can generate concordant results with current CE-based methods. In addition, MPS-based systems can facilitate mixture deconvolution with the detection of intra-repeat variations within length-based STR alleles. [ABSTRACT FROM AUTHOR]

Biological technology used to treat flue gas is useful to replace conventional treatment, but there is sulfide inhibition. However, no sulfide toxicity effect was observed in haloalkaliphilic bioreactors. The performance of the ethanol-fed bioreactor was better than that of lactate-, glucose-, and formate-fed bioreactor, respectively. To support this result strongly, Illumina MiSeq paired-end sequencing of 16S rRNA gene was applied to investigate the bacterial communities. A total of 389,971 effective sequences were obtained and all of them were assigned to 10,220 operational taxonomic units (OTUs) at a 97% similarity. Bacterial communities in the glucose-fed bioreactor showed the greatest richness and evenness. The highest relative abundance of sulfate-reducing bacteria (SRB) was found in the ethanol-fed bioreactor, which can explain why the performance of the ethanol-fed bioreactor was the best. Different types of SRB, sulfur-oxidizing bacteria, and sulfur-reducing bacteria were detected, indicating that sulfur may be cycled among these microorganisms. Because high-throughput 16S rRNA gene paired-end sequencing has improved resolution of bacterial community analysis, many rare microorganisms were detected, such as Halanaerobium , Halothiobacillus , Desulfonatronum , Syntrophobacter , and Fusibacter . 16S rRNA gene sequencing of these bacteria would provide more functional and phylogenetic information about the bacterial communities. [ABSTRACT FROM AUTHOR]

We developed microsatellite markers for genetic structural analyses of Dorcus hopei, a stag beetle species, using next generation sequencing and polymerase chain reaction (PCR)-based genotyping for regional populations. A total of 407,070,351 base pairs of genomic DNA containing >4000 microsatellite loci except AT repeats were sequenced. From 76 loci selected for primer design, 27 were polymorphic. Of these 27 markers, 10 were tested on three regional populations: two Chinese (Shichuan and Guangxi) and one Korean (Wanju). Three markers were excluded due to inconsistent amplification, genotyping errors, and Hardy-Weinberg equilibrium (HWE). By multi-locus genotyping, the allele number, observed heterozygosity and polymorphism information content of seven microsatellite loci were ranged 2-10, 0.1333-1.0000, and 0.1228-0.8509, respectively. In an analysis on the genetic differentiation among regional populations including one Japanese population and one cross-breeding population, the individual colored bar-plots showed that both Chinese populations were closer to each other than to the Far East Asian populations. In Far East Asian populations, Wanju and Nirasaki populations could not be distinguished from each other because the frequency of genetic contents was very similar in some individuals of two populations. Moreover, the cross-breeding population contained all patterns of genetic contents shown in Chinese, Korean, and Japanese populations, compared with the genetic content frequency of each regional population. As a result, we examined whether the cross-breeding population might be a hybrid population, and might contain a possibility of interbreeding with Chinese populations in parental generations. Therefore, these markers will be useful for analyses of genetic diversity in populations, genetic relationships between regional populations, genetic structure analyses, and origin tests. [ABSTRACT FROM AUTHOR]

A 23-mutation panel for CFTR carrier screening is recommended to women of reproductive age by the American College of Obstetricians and Gynecologists. In the present study the optimized efficiency regarding the carrier rate of Next-Generation sequencing (NGS) technology is compared to the one of limited mutation detection panels. A total of 824 consequent cases were subjected to the commercial Cystic Fibrosis Genotyping Assay. Some 188 negative samples randomly selected from the initial group of probands were further subjected to an extended mutation panel characterized by 92% detection rate, as well as to massive parallel sequencing. Twenty-two probands subjected to the commercial assay proved to carry one mutation included in the ACOG panel (carrier rate 0.0267). The latter panels revealed the presence of mutations not included in the ACOG panel in four probands, resulting to an increase of carrier rate of 0.0106 in the case of in-house panel and an increase of rate of 0.0213 if NGS was used. The above data seem to support the implementation of NGS in the routine CFTR carrier screening. [ABSTRACT FROM AUTHOR]

Recent obesity studies in humans and rodents have suggested that host weight gain is significantly associated with energy harvesting efficiency which is regulated by gut microbiota. Antibiotic growth promoters have been banned as feed additives in many countries. In this study, we aimed to provide knowledge of swine fecal microbiota by analyzing bacterial 16S rRNA gene sequences. Our results showed that swine fecal bacterial composition varied at each growth stage. Bacteroidetes decreased as the swine gained weight and unclassified genera significantly increased at later growth stages. Operational taxonomic unit (OTU) distribution analysis showed that the bacterial community difference was most significant between growers and finishers, while analysis of shared OTUs indicated a greater proportion of common species between growers and finishers. The differential abundance test between growers and finishers detected that nearly half of the species were shared OTUs, suggesting that differential abundance of each bacterial species predominantly controls bacterial community differences. Although functions of these bacteria are yet to be identified, understanding differences in fecal microbiota between each growth stage will provide additional insights for further studies related to swine gut microbiota. [ABSTRACT FROM AUTHOR]

Sequence-independent methods for viral discovery have been widely used for whole genome sequencing of viruses. Different protocols for viral enrichment, library preparation and sequencing have increasingly been more available and at lower costs. However, no study to date has focused on optimization of viral sample preparation for commercial deep sequencing. Accordingly, the aim of the present study was to evaluate an In-House enzymatic protocol for double-stranded DNA (dsDNA) synthesis and also compare the use of a commercially available kit protocol (Nextera XT, Illumina Inc, San Diego, CA, USA) and its combination with a library quantitation kit (Kapa, Kapa Biosystems, Wilmington, MA, USA) for deep sequencing (Illumina Miseq). Two RNA viruses (canine distemper virus and dengue virus) and one ssDNA virus (porcine circovirus type 2) were tested with the optimized protocols. The tested method for dsDNA synthesis has shown satisfactory results and may be used in laboratory setting, particularly when enzymes are already available. Library preparation combining commercial kits (Nextera XT and Kapa) has yielded more reads and genome coverage, probably due to a lack of small fragment recovering at the normalization step of Nextera XT. In addition, libraries may be diluted or concentrated to provide increase on genome coverage with Kapa quantitation. [ABSTRACT FROM AUTHOR]

The bacterial communities of activated sludge from nine wastewater treatment plants (WWTPs) located in three cities in China were examined using high-throughput MiSeq sequencing. The results showed that there were 57 genera of bacterial populations commonly shared by all nine systems, including Ferruginibacter, Dechloromonas, Zoogloea, Gp4, Gp6, etc., indicating that there was a core microbial community in the microbial populations of WWTPs at different geographic locations. Canonical correspondence analysis (CCA) results indicated that the bacterial community variance correlated most strongly with water temperature, dissolved oxygen (DO), concentrations of chemical oxygen demand (COD), and solids retention time (SRT). Variance partitioning analyses suggested that wastewater characteristics had the greatest contribution to the bacterial community variance, explaining 21% of the variance of bacterial communities independently, followed by operational parameters (17%), and geographic location (11%). This study provided insights into the diversity and bacterial community structures in geographically distributed WWTPs. [ABSTRACT FROM AUTHOR]

Use of complete mitochondrial genomes (mitogenomes) can greatly increase the resolution achievable in phylogeographic and historical demographic studies. Using next-generation sequencing methods, it is now feasible to efficiently sequence mitogenomes of large numbers of individuals once a reference mitogenome is available. However, assembling the initial mitogenomes of nonmodel organisms can present challenges, for example, in birds, where mtDNA is often subject to gene rearrangements and duplications. We developed a workflow based on Illumina paired-end, whole-genome shotgun sequencing, which we used to generate complete 19-kilobase mitogenomes for each of three species of North Pacific albatross, a group of birds known to carry a tandem duplication. Although this duplication had been described previously, our procedure did not depend on this prior knowledge, nor did it require a closely related reference mitogenome (e.g. a mammalian mitogenome was sufficient). We employed an iterative process including de novo assembly, reference-guided assembly and gap closing, which enabled us to detect duplications, determine gene order and identify sequence for primer positioning to resolve any mitogenome ambiguity (via minimal targeted Sanger sequencing). We present full mtDNA annotations, including 22 tRNAs, 2 rRNAs, 13 protein-coding genes, a control region and a duplicated feature for all three species. Pairwise comparisons supported previous hypotheses regarding the phylogenetic relationships within this group and occurrence of a shared tandem duplication. The resulting mitogenome sequences will enable rapid, high-throughput NGS mitogenome sequencing of North Pacific albatrosses via direct reference-guided assembly. Moreover, our approach to assembling mitogenomes should be applicable to any taxon. [ABSTRACT FROM AUTHOR]

High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology. [ABSTRACT FROM AUTHOR]

Background: The cystic fibrosis transmembrane conductance regulator ( CFTR) gene, responsible for the development of cystic fibrosis, is known as a pancreatitis susceptibility gene. Direct DNA sequencing of PCR-amplified CFTR gene segments is a first-line method to detect unknown mutations, but it is a tedious and labor-intensive endeavor given the large size of the gene (27 exons, 1,480 amino acids). Next-generation sequencing (NGS) is becoming standardized, reducing the cost of DNA sequencing, and enabling the generation of millions of reads per run. We here report a comprehensive analysis of CFTR variants in Japanese patients with chronic pancreatitis using NGS coupling with target capture. Methods: Exon sequences of the CFTR gene from 193 patients with chronic pancreatitis (121 idiopathic, 46 alcoholic, 17 hereditary, and nine familial) were captured by HaloPlex target enrichment technology, followed by NGS. Results: The sequencing data covered 91.6 % of the coding regions of the CFTR gene by ≥20 reads with a mean read depth of 449. We could identify 12 non-synonymous variants including three novel ones [c.A1231G (p.K411E), c.1753G>T (p.E585X) and c.2869delC (p.L957fs)] and seven synonymous variants including three novel ones in the exonic regions. The frequencies of the c.4056G>C (p.Q1352H) and the c.3468G>T (p.L1156F) variants were higher in patients with chronic pancreatitis than those in controls. Conclusions: Target sequence capture combined with NGS is an effective method for the analysis of pancreatitis susceptibility genes. [ABSTRACT FROM AUTHOR]

High-throughput sequencing platforms are continuing to increase resulting read lengths, which is allowing for a deeper and more accurate depiction of environmental microbial diversity. With the nascent Reagent Kit v3, Illumina MiSeq now has the ability to sequence the eukaryotic hyper-variable V4 region of the SSU-r DNA locus with paired-end reads. Using DNA collected from soils with analyses of strictly- and nearly identical amplicons, here we ask how the new Illumina MiSeq data compares with what we can obtain with Roche/454 GS FLX with regard to quantity and quality, presence and absence, and abundance perspectives. We show that there is an easy qualitative transition from the Roche/454 to the Illumina MiSeq platforms. The ease of this transition is more nuanced quantitatively for low-abundant amplicons, although estimates of abundances are known to also vary within platforms. [ABSTRACT FROM AUTHOR]

Microsatellite instability (MSI) is a useful marker for risk assessment, prediction of chemotherapy responsiveness and prognosis in patients with colorectal cancer. Here, we describe a next generation sequencing approach for MSI testing using the MiSeq platform. Different from other MSI capturing strategies that are based on targeted gene capture, we utilize "deep resequencing", where we focus the sequencing on only the microsatellite regions of interest. We sequenced a series of 44 colorectal tumours with normal controls for five MSI loci (BAT25, BAT26, BAT34c4, D18S55, D5S346) and a second series of six colorectal tumours (no control) with two mononucleotide loci (BAT25, BAT26). In the first series, we were able to determine 17 MSI-High, 1 MSI-Low and 26 microsatellite stable (MSS) tumours. In the second series, there were three MSI-High and three MSS tumours. Although there was some variation within individual markers, this NGS method produced the same overall MSI status for each tumour, as obtained with the traditional multiplex PCR-based method. [ABSTRACT FROM AUTHOR]

Routine use of massively parallel sequencing (MPS) for forensic genomics is on the horizon. The last few years, several algorithms and workflows have been developed to analyze forensic MPS data. However, none have yet been tailored to the needs of the forensic analyst who does not possess an extensive bioinformatics background. We developed our previously published forensic MPS data analysis framework MyFLq (My-Forensic-Loci-queries) into an open-source, user-friendly, web-based application. It can be installed as a standalone web application, or run directly from the Illumina BaseSpace environment. In the former, laboratories can keep their data on-site, while in the latter, data from forensic samples that are sequenced on an Illumina sequencer can be uploaded to Basespace during acquisition, and can subsequently be analyzed using the published MyFLq BaseSpace application. Additional features were implemented such as an interactive graphical report of the results, an interactive threshold selection bar, and an allele length-based analysis in addition to the sequenced-based analysis. Practical use of the application is demonstrated through the analysis of four 16-plex short tandem repeat (STR) samples, showing the complementarity between the sequence- and length-based analysis of the same MPS data. [ABSTRACT FROM AUTHOR]

The cost of DNA sequencing has decreased due to advancements in Next Generation Sequencing. The number of sequences obtained from the Illumina platform is large, use of this platform can reduce costs more than the 454 pyrosequencer. However, the Illumina platform has other challenges, including bioinformatics analysis of large numbers of sequences and the need to reduce erroneous nucleotides generated at the 3′-ends of the sequences. These erroneous sequences can lead to errors in analysis of microbial communities. Therefore, correction of these erroneous sequences is necessary for accurate taxonomic identification. Several studies that have used the Illumina platform to perform metagenomic analyses proposed curating pipelines to increase accuracy. In this study, we evaluated the likelihood of obtaining an erroneous microbial composition using the MiSeq 250 bp paired sequence platform and improved the pipeline to reduce erroneous identifications. We compared different sequencing conditions by varying the percentage of control phiX added, the concentration of the sequencing library, and the 16S rRNA gene target region using a mock community sample composed of known sequences. Our recommended method corrected erroneous nucleotides and improved identification accuracy. Overall, 99.5% of the total reads shared 95% similarity with the corresponding template sequences and 93.6% of the total reads shared over 97% similarity. This indicated that the MiSeq platform can be used to analyze microbial communities at the genus level with high accuracy. The improved analysis method recommended in this study can be applied to amplicon studies in various environments using high-throughput reads generated on the MiSeq platform. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genomes of four fish species of the commercially important family Latidae were sequenced using the Illumina MiSeq, thereby significantly increasing the mitogenomic resources for the family. Whole mitogenome-based phylogenetic analysis supports the monophyly of the genus Lates and more generally the family Latidae. The mitogenome sequences from this study will be useful for future assessments of the diversity within and between Lates species and studies of phylogenetic relationships within the diverse and taxonomically challenging perciform fishes. [ABSTRACT FROM AUTHOR]

A greenhouse experiment was conducted to investigate the effects of zucchini ( Cucurbita pepo L.), inoculated with the arbuscular mycorrhizal (AM) species Acaulospora laevis, Glomus caledonium, and Glomus mosseae, on the soil bacterial community responsible for Aroclor 1242 dissipation. The dissipation rates of Aroclor 1242 and soil bacteria abundance were much higher with the A. laevis and G. mosseae treatments compared to the non-mycorrhizal control. The biphenyl dioxygenase ( bphA) and Rhodococcus-like 2,3-dihydroxybiphenyl dioxygenase ( bphC) genes were more abundant in AM inoculated soils, suggesting that the bphA and Rhodococcus-like bphC pathways play an important role in Aroclor 1242 dissipation in the mycorrhizosphere. The soil bacterial communities were dominated by classes Betaproteobacteria and Actinobacteria, while the relative proportion of Actinobacteria was significantly ( F = 2.288, P < 0.05) correlated with the PCB congener profile in bulk soil. Our results showed that AM fungi could enhance PCB dissipation by stimulating bph gene abundance and the growth of specific bacterial groups. [ABSTRACT FROM AUTHOR]

Biodiversity, phylogeography and population genetic studies will be revolutionized by access to large data sets thanks to next-generation sequencing methods. In this study, we develop an easy and cost-effective protocol for in-solution enrichment hybridization capture of complete chloroplast genomes applicable at deep-multiplexed levels. The protocol uses cheap in-house species-specific probes developed via long-range PCR of the entire chloroplast. Barcoded libraries are constructed, and in-solution enrichment of the chloroplasts is carried out using the probes. This protocol was tested and validated on six economically important West African crop species, namely African rice, pearl millet, three African yam species and fonio. For pearl millet, we also demonstrate the effectiveness of this protocol to retrieve 95% of the sequence of the whole chloroplast on 95 multiplexed individuals in a single MiSeq run at a success rate of 95%. This new protocol allows whole chloroplast genomes to be retrieved at a modest cost and will allow unprecedented resolution for closely related species in phylogeography studies using plastomes. [ABSTRACT FROM AUTHOR]

The development of molecular tools to detect and report mitochondrial DNA (mtDNA) heteroplasmy will increase the discrimination potential of the testing method when applied to forensic cases. The inherent limitations of the current state-of-the-art, Sanger-based sequencing, including constrictions in speed, throughput, and resolution, have hindered progress in this area. With the advent of next-generation sequencing (NGS) approaches, it is now possible to clearly identify heteroplasmic variants, and at a much lower level than previously possible. However, in order to bring these approaches into forensic laboratories and subsequently as accepted scientific information in a court of law, validated methods will be required to produce and analyze NGS data. We report here on the development of an optimized approach to NGS analysis for the mtDNA genome (mtgenome) using the Illumina MiSeq instrument. This optimized protocol allows for the production of more than 5 gigabases of mtDNA sequence per run, sufficient for detection and reliable reporting of minor heteroplasmic variants down to approximately 0.5–1.0% when multiplexing twelve samples. Depending on sample throughput needs, sequence coverage rates can be set at various levels, but were optimized here for at least 5000 reads. In addition, analysis parameters are provided for a commercially available software package that identify the highest quality sequencing reads and effectively filter out sequencing-based noise. With this method it will be possible to measure the rates of low-level heteroplasmy across the mtgenome, evaluate the transmission of heteroplasmy between the generations of maternal lineages, and assess the drift of variant sequences between different tissue types within an individual. [ABSTRACT FROM AUTHOR]

Complete mitochondrial genomes have been shown to be reliable markers for phylogeny reconstruction among diverse animal groups. However, the relative difficulty and high cost associated with obtaining de novo full mitogenomes have frequently led to conspicuously low taxon sampling in ensuing studies. Here, we report the successful use of an economical and accessible method for assembling complete or near-complete mitogenomes through shot-gun next-generation sequencing of a single library made from pooled total DNA extracts of numerous target species. To avoid the use of separate indexed libraries for each specimen, and an associated increase in cost, we incorporate standard polymerase chain reaction-based “bait” sequences to identify the assembled mitogenomes. The method was applied to study the higher level phylogenetic relationships in the weevils (Coleoptera: Curculionoidea), producing 92 newly assembled mitogenomes obtained in a single Illumina MiSeq run. The analysis supported a separate origin of wood-boring behavior by the subfamilies Scolytinae, Platypodinae, and Cossoninae. This finding contradicts morphological hypotheses proposing a close relationship between the first two of these but is congruent with previous molecular studies, reinforcing the utility of mitogenomes in phylogeny reconstruction. Our methodology provides a technically simple procedure for generating densely sampled trees from whole mitogenomes and is widely applicable to groups of animals for which bait sequences are the only required prior genome knowledge. [ABSTRACT FROM PUBLISHER]

Background: Next-generation sequencers (NGSs) have become one of the main tools for current biology. To obtain useful insights from the NGS data, it is essential to control low-quality portions of the data affected by technical errors such as air bubbles in sequencing fluidics. Results: We develop a software SUGAR (subtile-based GUI-assisted refiner) which can handle ultra-high-throughput data with user-friendly graphical user interface (GUI) and interactive analysis capability. The SUGAR generates high-resolution quality heatmaps of the flowcell, enabling users to find possible signals of technical errors during the sequencing. The sequencing data generated from the error-affected regions of a flowcell can be selectively removed by automated analysis or GUI-assisted operations implemented in the SUGAR. The automated data-cleaning function based on sequence read quality (Phred) scores was applied to a public whole human genome sequencing data and we proved the overall mapping quality was improved. Conclusion: The detailed data evaluation and cleaning enabled by SUGAR would reduce technical problems in sequence read mapping, improving subsequent variant analysis that require high-quality sequence data and mapping results. Therefore, the software will be especially useful to control the quality of variant calls to the low population cells, e.g., cancers, in a sample with technical errors of sequencing procedures. [ABSTRACT FROM AUTHOR]

Objectives: We developed a new human papillomavirus (HPV) genotyping assay based on multiplex polymerase chain reaction and next-generation sequencing (NGS) methods for large-scale cervical cancer screening. Methods: We first trained the assay on 1,170 self-collected samples, balancing the cutoff points for high-risk types. Then using 4,262 separate self-collected specimens, we compared concordance, sensitivity, and specificity for cervical intraepithelial neoplasia type 2 (CIN2) or higher and CIN type 3 (CIN3) or higher of the HPV sequencing assay with that of Hybrid Capture 2 (HC2) direct samples and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay self-samples. Results: All assays had a good agreement. The sensitivity for CIN2 or higher and CIN3 or higher of the self-sampling specimens tested with the sequencing assay run on both MiSeq and Ion Torrent Personal Genome Machine sequencer was similar to that of direct-sampling specimens tested with HC2 (P > .05), but the specificity of the sequencing assay for CIN2 or higher and CIN3 or higher was significantly higher than that of HC2 (P < .01). Conclusions: This population-based study has demonstrated the applicability of a new NGS high-risk HPV assay for primary cervical cancer screening based on self-collection. [ABSTRACT FROM AUTHOR]