Combination Of Next-generation Sequencing Research Articles

Whole-exome sequencing and imaging genetics identify functional variants for rate of change in hippocampal volume in mild cognitive impairment

Whole-exome sequencing of individuals with mild cognitive impairment, combined with genotype imputation, was used to identify coding variants other than the apolipoprotein E (APOE) ε4 allele associated with rate of hippocampal volume loss using an extreme trait design. Matched unrelated APOE ε3 homozygous male Caucasian participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI) were selected at the extremes of the 2-year longitudinal change distribution of hippocampal volume (eight subjects with rapid rates of atrophy and eight with slow/stable rates of atrophy). We identified 57 non-synonymous single nucleotide variants (SNVs) which were found exclusively in at least 4 of 8 subjects in the rapid atrophy group, but not in any of the 8 subjects in the slow atrophy group. Among these SNVs, the variants that accounted for the greatest group difference and were predicted in silico as 'probably damaging' missense variants were rs9610775 (CARD10) and rs1136410 (PARP1). To further investigate and extend the exome findings in a larger sample, we conducted quantitative trait analysis including whole-brain search in the remaining ADNI APOE ε3/ε3 group (N=315). Genetic variation within PARP1 and CARD10 was associated with rate of hippocampal neurodegeneration in APOE ε3/ε3. Meta-analysis across five independent cross sectional cohorts indicated that rs1136410 is also significantly associated with hippocampal volume in APOE ε3/ε3 individuals (N=923). Larger sequencing studies and longitudinal follow-up are needed for confirmation. The combination of next-generation sequencing and quantitative imaging phenotypes holds significant promise for discovery of variants involved in neurodegeneration.

Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.

Phylogenetic and functional metagenomic profiling for assessing microbial biodiversity in environmental monitoring.

Decisions guiding environmental management need to be based on a broad and comprehensive understanding of the biodiversity and functional capability within ecosystems. Microbes are of particular importance since they drive biogeochemical cycles, being both producers and decomposers. Their quick and direct responses to changes in environmental conditions modulate the ecosystem accordingly, thus providing a sensitive readout. Here we have used direct sequencing of total DNA from water samples to compare the microbial communities of two distinct coastal regions exposed to different anthropogenic pressures: the highly polluted Port of Genoa and the protected area of Montecristo Island in the Mediterranean Sea. Analysis of the metagenomes revealed significant differences in both microbial diversity and abundance between the two areas, reflecting their distinct ecological habitats and anthropogenic stress conditions. Our results indicate that the combination of next generation sequencing (NGS) technologies and bioinformatics tools presents a new approach to monitor the diversity and the ecological status of aquatic ecosystems. Integration of metagenomics into environmental monitoring campaigns should enable the impact of the anthropogenic pressure on microbial biodiversity in various ecosystems to be better assessed and also predicted.

Discovery of Novel MicroRNAs in Rat Kidney Using Next Generation Sequencing and Microarray Validation

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. The latest version of the miRBase database (Release 18) includes 1,157 mouse and 680 rat mature miRNAs. Only one new rat mature miRNA was added to the rat miRNA database from version 16 to version 18 of miRBase, suggesting that many rat miRNAs remain to be discovered. Given the importance of rat as a model organism, discovery of the completed set of rat miRNAs is necessary for understanding rat miRNA regulation. In this study, next generation sequencing (NGS), microarray analysis and bioinformatics technologies were applied to discover novel miRNAs in rat kidneys. MiRanalyzer was utilized to analyze the sequences of the small RNAs generated from NGS analysis of rat kidney samples. Hundreds of novel miRNA candidates were examined according to the mappings of their reads to the rat genome, presence of sequences that can form a miRNA hairpin structure around the mapped locations, Dicer cleavage patterns, and the levels of their expression determined by both NGS and microarray analyses. Nine novel rat hairpin precursor miRNAs (pre-miRNA) were discovered with high confidence. Five of the novel pre-miRNAs are also reported in other species while four of them are rat specific. In summary, 9 novel pre-miRNAs (14 novel mature miRNAs) were identified via combination of NGS, microarray and bioinformatics high-throughput technologies.

Identification of a novel nidovirus associated with a neurological disease of the Australian brushtail possum (Trichosurus vulpecula)

A novel, fatal neurological disease of the Australian brushtail possum (Trichosurus vulpecula) was first identified in 1995 in a research facility and subsequently in free-living possums in New Zealand and termed wobbly possum disease (WPD). The results of previous transmission studies suggested that the aetiological agent of WPD is most likely a virus. However, the identity of the presumed viral agent had not been elucidated. In the current report, we describe identification of a novel virus from tissues of WPD-affected possums using a combination of next generation sequencing and traditional molecular methods. The proportion of possums positive for the novel virus by PCR was significantly higher (p<0.0001) among animals with WPD than clinically healthy possums, strongly suggesting an aetiological involvement of the virus in WPD. Analysis of the partial genomic sequence of the putative WPD virus indicated that it is a novel nidovirus, most closely related to the current members of the family Arteriviridae.

Mutations in the Alpha 1,2-Mannosidase Gene, MAN1B1, Cause Autosomal-Recessive Intellectual Disability

We have used genome-wide genotyping to identify an overlapping homozygosity-by-descent locus on chromosome 9q34.3 (MRT15) in four consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability (NS-ARID) and one in which the patients show additional clinical features. Four of the families are from Pakistan, and one is from Iran. Using a combination of next-generation sequencing and Sanger sequencing, we have identified mutations in the gene MAN1B1, encoding a mannosyl oligosaccharide, alpha 1,2-mannosidase. In one Pakistani family, MR43, a homozygous nonsense mutation (RefSeq number NM_016219.3: c.1418G>A [p.Trp473*]), segregated with intellectual disability and additional dysmorphic features. We also identified the missense mutation c. 1189G>A (p.Glu397Lys; RefSeq number NM_016219.3), which segregates with NS-ARID in three families who come from the same village and probably have shared inheritance. In the Iranian family, the missense mutation c.1000C>T (p.Arg334Cys; RefSeq number NM_016219.3) also segregates with NS-ARID. Both missense mutations are at amino acid residues that are conserved across the animal kingdom, and they either reduce k(cat) by ∼1300-fold or disrupt stable protein expression in mammalian cells. MAN1B1 is one of the few NS-ARID genes with an elevated mutation frequency in patients with NS-ARID from different populations.