Low-pass genome-wide sequencing and variant inference using identity-by-descent in an isolated human population

Whole-genome sequencing in an isolated population with few founders directly ascertains variants from the population bottleneck that may be rare elsewhere. In such populations, shared haplotypes allow imputation of variants in unsequenced samples without resorting to complex statistical methods as in studies of outbred cohorts. We focus on an isolated population cohort from the Pacific Island of Kosrae, Micronesia, where we previously collected SNP array and rich phenotype data for the majority of the population. We report identification of long regions with haplotypes co-inherited between pairs of individuals and methodology to leverage such shared genetic content for imputation. Our estimates show that sequencing as few as 40 personal genomes allows for inference in up to 60% of the 3000-person cohort at the average locus. We ascertained a pilot data set of whole-genome sequences from seven Kosraean individuals, with average 5× coverage. This assay identified 5,735,306 unique sites of which 1,212,831 were previously unknown. Additionally, these variants are unusually enriched for alleles that are rare in other populations when compared to geographic neighbors (published Korean genome SJK). We used the presence of shared haplotypes between the seven Kosraen individuals to estimate expected imputation accuracy of known and novel homozygous variants at 99.6% and 97.3%, respectively. This study presents whole-genome analysis of a homogenous isolate population with emphasis on optimal rare variant inference.     

Highly accurate-single chromosomal complete genomes using IonTorrent and MinION sequencing of clinical pathogens

Oxford Nanopore MinION sequencing technology has been gaining immense importance in identification of pathogen and antimicrobial resistance, though with 10–15% error rate. Short read technologies generates high accurate genome but with multiple fragments of genome. This study proposes a novel workflow to reduce the indels resulted from MinION long read sequencing by overlaying short read sequences from IonTorrent in the clinical isolates. Best of both techniques were employed which generated highly accurate-single chromosomal microbial genomes with increase in completeness of genomes from 44.5%, 30% and 43% to 98.6%, 98.6% and 96.6% for P. aeruginosa, A. veronii and B. pertussis respectively. To the best of our knowledge, this is the first study to generate a hybrid of IonTorrent and MinION reads to obtain single chromosomal genomes. This would enable to precisely infer both structural arrangement of genes and SNP based analysis for phylogenetic information.     

A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits

The enrichment of targeted regions within complex next generation sequencing libraries commonly uses biotinylated baits to capture the desired sequences. This method results in high read coverage over the targets and their flanking regions. Oxford Nanopore Technologies recently released an USB3.0-interfaced sequencer, the MinION. To date no particular method for enriching MinION libraries has been standardized. Here, using biotinylated PCR-generated baits in a novel approach, we describe a simple and efficient way for multiplexed enrichment of MinION libraries, overcoming technical limitations related with the chemistry of the sequencing-adapters and the length of the DNA fragments. Using Phage Lambda and Escherichia coli as models we selectively enrich for specific targets, significantly increasing the corresponding read-coverage, eliminating unwanted regions. We show that by capturing genomic fragments, which contain the target sequences, we recover reads extending targeted regions and thus can be used for the determination of potentially unknown flanking sequences. By pooling enriched libraries derived from two distinct E. coli strains and analyzing them in parallel, we demonstrate the efficiency of this method in multiplexed format. Crucially we evaluated the optimal bait size for large fragment libraries and we describe for the first time a standardized method for target enrichment in MinION platform.     

Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

Background

Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. We compared the suitability of two benchtop next-generation sequencers for whole genome influenza A quasispecies analysis: the Illumina MiSeq sequencing-by-synthesis and the Ion Torrent PGM semiconductor sequencing technique.

Results

We first compared the accuracy and sensitivity of both sequencers using plasmid DNA and different ratios of wild type and mutant plasmid. Illumina MiSeq sequencing reads were one and a half times more accurate than those of the Ion Torrent PGM. The majority of sequencing errors were substitutions on the Illumina MiSeq and insertions and deletions, mostly in homopolymer regions, on the Ion Torrent PGM. To evaluate the suitability of the two techniques for determining the genome diversity of influenza A virus, we generated plasmid-derived PR8 virus and grew this virus in vitro. We also optimized an RT-PCR protocol to obtain uniform coverage of all eight genomic RNA segments. The sequencing reads obtained with both sequencers could successfully be assembled de novo into the segmented influenza virus genome. After mapping of the reads to the reference genome, we found that the detection limit for reliable recognition of variants in the viral genome required a frequency of 0.5% or higher. This threshold exceeds the background error rate resulting from the RT-PCR reaction and the sequencing method. Most of the variants in the PR8 virus genome were present in hemagglutinin, and these mutations were detected by both sequencers.

Conclusions

Our approach underlines the power and limitations of two commonly used next-generation sequencers for the analysis of influenza virus gene diversity. We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time. The data analysis pipeline that we propose here will also help to standardize variant calling in small RNA genomes based on next-generation sequencing data.     

A novel genome-information content-based statistic for genome-wide association analysis designed for next-generation sequencing data

The genome-wide association studies (GWAS) designed for next-generation sequencing data involve testing association of genomic variants, including common, low frequency, and rare variants. The current strategies for association studies are well developed for identifying association of common variants with the common diseases, but may be ill-suited when large amounts of allelic heterogeneity are present in sequence data. Recently, group tests that analyze their collective frequency differences between cases and controls shift the current variant-by-variant analysis paradigm for GWAS of common variants to the collective test of multiple variants in the association analysis of rare variants. However, group tests ignore differences in genetic effects among SNPs at different genomic locations. As an alternative to group tests, we developed a novel genome-information content-based statistics for testing association of the entire allele frequency spectrum of genomic variation with the diseases. To evaluate the performance of the proposed statistics, we use large-scale simulations based on whole genome low coverage pilot data in the 1000 Genomes Project to calculate the type 1 error rates and power of seven alternative statistics: a genome-information content-based statistic, the generalized T(2), collapsing method, multivariate and collapsing (CMC) method, individual χ(2) test, weighted-sum statistic, and variable threshold statistic. Finally, we apply the seven statistics to published resequencing dataset from ANGPTL3, ANGPTL4, ANGPTL5, and ANGPTL6 genes in the Dallas Heart Study. We report that the genome-information content-based statistic has significantly improved type 1 error rates and higher power than the other six statistics in both simulated and empirical datasets.     

Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis

Mutation mapping in mice can be readily accomplished by genome wide segregation analysis of polymorphic DNA markers. In this study, we showed the efficacy of Ion Torrent next generation sequencing for conducting genome-wide scans to map and identify a mutation causing congenital heart disease in a mouse mutant, Bishu, recovered from a mouse mutagenesis screen. The Bishu mutant line generated in a C57BL/6J (B6) background was intercrossed with another inbred strain, C57BL/10J (B10), and the resulting B6/B10 hybrid offspring were intercrossed to generate mutants used for the mapping analysis. For each mutant sample, a panel of 123 B6/B10 polymorphic SNPs distributed throughout the mouse genome was PCR amplified, bar coded, and then pooled to generate a single library used for Ion Torrent sequencing. Sequencing carried out using the 314 chip yielded >600,000 usable reads. These were aligned and mapped using a custom bioinformatics pipeline. Each SNP was sequenced to a depth >500×, allowing accurate automated calling of the B6/B10 genotypes. This analysis mapped the mutation in Bishu to an interval on the proximal region of mouse chromosome 4. This was confirmed by parallel capillary sequencing of the 123 polymorphic SNPs. Further analysis of genes in the map interval identified a splicing mutation in Dnaic1 ^c.204+1G>A, an intermediate chain dynein, as the disease causing mutation in Bishu. Overall, our experience shows Ion Torrent amplicon sequencing is high throughput and cost effective for conducting genome-wide mapping analysis and is easily scalable for other high volume genotyping analyses.     

A genome-wide gain-of-function analysis of rice genes using the FOX-hunting system

The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.     

Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries

Insertion sequencing (INSeq) is a method for determining the insertion site and relative abundance of large numbers of transposon mutants in a mixed population of isogenic mutants of a sequenced microbial species. INSeq is based on a modified mariner transposon containing MmeI sites at its ends, allowing cleavage at chromosomal sites 16-17 bp from the inserted transposon. Genomic regions adjacent to the transposons are amplified by linear PCR with a biotinylated primer. Products are bound to magnetic beads, digested with MmeI and barcoded with sample-specific linkers appended to each restriction fragment. After limited PCR amplification, fragments are sequenced using a high-throughput instrument. The sequence of each read can be used to map the location of a transposon in the genome. Read count measures the relative abundance of that mutant in the population. Solid-phase library preparation makes this protocol rapid (18 h), easy to scale up, amenable to automation and useful for a variety of samples. A protocol for characterizing libraries of transposon mutant strains clonally arrayed in a multiwell format is provided.     

A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli

A simple procedure has been developed for sequencing long fragments of DNA. The fragment (which can be several kb in length) is cloned in pAA3.7X, and subdivided into many overlapping segments by Tn9-promoted deletions. The deletions are isolated by positive selection for galactose resistance. A rapid plasmid preparation from several hundred galactose-resistant colonies is fractionated by agarose gel electrophoresis to pick a series of deletions terminating at approx. 200-bp intervals across the entire length of the fragment. Selected plasmids are purified by rapid alkaline extraction, and used directly for supercoil sequencing with a primer derived from IS1. Sequences of adjacent deletions contain overlaps which are used to connect individual sequences to give the complete sequence.     

Whole-genome sequencing and phylogenetic analysis of rabies viruses from Jordan

Human fatalities caused by rabies are rarely reported in Jordan; however, domestic animals are more likely to fall victim to rabies compared to wild animals, at least this is the case in Jordan due to the presence of canine rabies. In this study, twelve brain samples from domestic and wild animals suspected of being infected with rabies virus from different regions of Jordan were collected during 2019. Seven of them tested positive using the fluorescent antibody test and real-time SYBR RT-PCR assay. Five specimens were from stray dogs and two from foxes. The whole genome sequences were obtained from the positive samples. Sequence analysis showed that one dog virus from Al Quwaysimah city located in Amman governorate, was closely related to an Israeli strain belonging to a Cosmopolitan ME1a clade. The genomes of the remaining six viruses (four from dogs and two from foxes) collected from different areas of Jordan were genetically-related to each other and clustered together with sequences from Iran and Turkey; all belong to Cosmopolitan ME2 clade. These sequences were analyzed with six other Jordanian rabies virus nucleoprotein (N) gene sequences available in the public database, five of them belong to ME1a clade and one belongs to ME1b clade. Rabies virus whole genome data is scarce across the Middle East. This study provides a better understanding of the molecular epidemiology of rabies virus in the region.     

Characterizing dopaminergic neuron vulnerability using genome-wide analysis

Parkinson’s disease (PD) is primarily characterized by the loss of dopaminergic (DA) neurons in the brain. However, little is known about why DA neurons are selectively vulnerable to PD. To identify genes that are associated with DA neuron loss, we screened through 201 wild-caught populations of Drosophila melanogaster as part of the Drosophila Genetic Reference Panel. Here, we identify the top-associated genes containing single-nucleotide polymorphisms that render DA neurons vulnerable. These genes were further analyzed by using mutant analysis and tissue-specific knockdown for functional validation. We found that this loss of DA neurons caused progressive locomotor dysfunction in mutants and gene knockdown analysis. The identification of genes associated with the progressive loss of DA neurons should help to uncover factors that render these neurons vulnerable in PD, and possibly develop strategies to make these neurons more resilient.     

Semiconductor-based sequencing of genome-wide DNA methylation states

Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we developed a MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is low cost, rapid, and scalable. We applied this protocol to demonstrate MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450 BeadChip array, and accurately identifies sites of differential DNA methylation. Furthermore, we applied an integrative approach to further investigate and confirm the role of DNA methylation in alternative splicing and to profile 5mC and 5hmC variants of DNA methylation in normal human brain tissue that is localized over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions.     

Dissecting the genotype in syndromic intellectual disability using whole exome sequencing in addition to genome-wide copy number analysis

When a known microimbalance affecting multiple genes is detected in a patient with syndromic intellectual disability, it is usually presumed causative for all observed features. Whole exome sequencing (WES) allows questioning this assumption. In this study of three families with children affected by unexplained syndromic intellectual disability, genome-wide copy number and subsequent analyses revealed a de novo maternal 1.1 Mb microdeletion in the 14q32 imprinted region causing a paternal UPD(14)-like phenotype, and two inherited 22q11.21 microduplications of 2.5 or 2.8 Mb. In patient 1 carrying the 14q32 microdeletion, tall stature and renal malformation were unexplained by paternal UPD(14), and there was no altered DLK1 expression or unexpected methylation status. By WES and filtering with a mining tool, a novel FBN1 missense variant was found in patient 1 and his mother, who both showed clinical features of Marfan syndrome by thorough anthropometric assessment, and a novel EYA1 missense variant as a probable cause of the renal malformation in the patient. In patient 2 with the 22q11.21 microduplication syndrome, skin hypo- and hyperpigmentation and two malignancies were only partially explained. By WES, compound heterozygous BLM stop founder mutations were detected causing Bloom syndrome. In male patient 3 carrying a 22q11.21 microduplication inherited from his unaffected father, WES identified a novel missense variant in the OPHN1 X-linked intellectual disability gene inherited from the unaffected mother as a possible additional cause for developmental delay. Thus, WES seems warranted in patients carrying microdeletions or microduplications, who have unexplained clinical features or microimbalances inherited from an unaffected parent.     

A rapid analysis of copepod feeding using FlowCAM

This study addressed the usefulness and reliability of usinga new plankton image analyzer, FlowCAM, for rapid analysis ofcopepod feeding by comparison with the conventional microscopicanalysis. We carried out bottle incubation experiments withtwo copepod species in the Oyashio region and analyzed the preyabundance prior to and after the incubation with a FlowCAM.From the volume-specific fluorescence intensity of particles,the FlowCAM successfully distinguished between zooplankton andphytoplankton and allowed an adequate evaluation of the copepodfeeding on zooplankton and phytoplankton. The analysis timefor one plankton sample was about 10 min, which was less thanone-tenth of the time required for microscopic enumeration.The FlowCAM is considered to be an efficient tool for rapidanalysis of copepod feeding particularly in studies of omnivory.     

A rapid generalized least squares model for a genome-wide quantitative trait association analysis in families

Genome-wide association studies (GWAS) using family data involve association analyses between hundreds of thousands of markers and a trait for a large number of related individuals. The correlations among relatives bring statistical and computational challenges when performing these large-scale association analyses. Recently, several rapid methods accounting for both within- and between-family variation have been proposed. However, these techniques mostly model the phenotypic similarities in terms of genetic relatedness. The familial resemblances in many family-based studies such as twin studies are not only due to the genetic relatedness, but also derive from shared environmental effects and assortative mating. In this paper, we propose 2 generalized least squares (GLS) models for rapid association analysis of family-based GWAS, which accommodate both genetic and environmental contributions to familial resemblance. In our first model, we estimated the joint genetic and environmental variations. In our second model, we estimated the genetic and environmental components separately. Through simulation studies, we demonstrated that our proposed approaches are more powerful and computationally efficient than a number of existing methods are. We show that estimating the residual variance-covariance matrix in the GLS models without SNP effects does not lead to an appreciable bias in the p values as long as the SNP effect is small (i.e. accounting for no more than 1% of trait variance).     

A genome-wide survey on basic helix-loop-helix transcription factors in giant panda

The giant panda (Ailuropoda melanoleuca) is a critically endangered mammalian species. Studies on functions of regulatory proteins involved in developmental processes would facilitate understanding of specific behavior in giant panda. The basic helix-loop-helix (bHLH) proteins play essential roles in a wide range of developmental processes in higher organisms. bHLH family members have been identified in over 20 organisms, including fruit fly, zebrafish, mouse and human. Our present study identified 107 bHLH family members being encoded in giant panda genome. Phylogenetic analyses revealed that they belong to 44 bHLH families with 46, 25, 15, 4, 11 and 3 members in group A, B, C, D, E and F, respectively, while the remaining 3 members were assigned into "orphan". Compared to mouse, the giant panda does not encode seven bHLH proteins namely Beta3a, Mesp2, Sclerax, S-Myc, Hes5 (or Hes6), EBF4 and Orphan 1. These results provide useful background information for future studies on structure and function of bHLH proteins in the regulation of giant panda development.