miRTRAP, a computational method for the systematic identification of miRNAs from high throughput sequencing data

MicroRNAs (miRs) have been broadly implicated in animal development and disease. We developed a novel computational strategy for the systematic, whole-genome identification of miRs from high throughput sequencing information. This method, miRTRAP, incorporates the mechanisms of miR biogenesis and includes additional criteria regarding the prevalence and quality of small RNAs arising from the antisense strand and neighboring loci. This program was applied to the simple chordate Ciona intestinalis and identified nearly 400 putative miR loci.     

Identification of candidate genes in rice for resistance to sheath blight disease by whole genome sequencing

Recent advances in whole genome sequencing (WGS) have allowed identification of genes for disease susceptibility in humans. The objective of our research was to exploit whole genome sequences of 13 rice (Oryza sativa L.) inbred lines to identify non-synonymous SNPs (nsSNPs) and candidate genes for resistance to sheath blight, a disease of worldwide significance. WGS by the Illumina GA IIx platform produced an average 5× coverage with ~700 K variants detected per line when compared to the Nipponbare reference genome. Two filtering strategies were developed to identify nsSNPs between two groups of known resistant and susceptible lines. A total of 333 nsSNPs detected in the resistant lines were absent in the susceptible group. Selected variants associated with resistance were found in 11 of 12 chromosomes. More than 200 genes with selected nsSNPs were assigned to 42 categories based on gene family/gene ontology. Several candidate genes belonged to families reported in previous studies, and three new regions with novel candidates were also identified. A subset of 24 nsSNPs detected in 23 genes was selected for further study. Individual alleles of the 24 nsSNPs were evaluated by PCR whose presence or absence corresponded to known resistant or susceptible phenotypes of nine additional lines. Sanger sequencing confirmed presence of 12 selected nsSNPs in two lines. “Resistant” nsSNP alleles were detected in two accessions of O. nivara that suggests sources for resistance occur in additional Oryza sp. Results from this study provide a foundation for future basic research and marker-assisted breeding of rice for sheath blight resistance.     

Identification of a novel candidate gene for rolled leaf in rice

A semi-narrow and adaxially rolled leaf mutant, rl15(t), was induced from Korean japonica rice cultivar Ilpum by chemical mutagenesis using ethyl methanesulfonate. We characterized the mutant and identified the novel gene causing the mutant phenotype. Cytological analysis of mutant leaves indicated that the adaxial leaf-rolling phenotype is due to the reduced size and number of bulliform cells in the mutant. Genetic analysis showed that the rolled leaf trait is controlled by a single recessive gene, designated rl15(t). Using an F₂ mapping population generated from a cross between Milyang23 and the mutant, we mapped the candidate region to a 174 kb interval on the long arm of chromosome 1 near the centromeric region. Through whole genome sequencing in bulk and MutMap analysis, we identified the causal SNP within the candidate region. The results of RT-PCR analysis indicated that a splicing error occurred due to a base change from G to A at the beginning of the fifth intron of LOC_Os01g37837, which encodes a putative seryl-tRNA synthetase, resulting in the mutant phenotype. Further study of the rl15(t) gene will facilitate analysis of leaf architecture and morphogenesis in rice plants.     

Identification of novel candidate genes by exome sequencing in Tunisian familial male breast cancer patients

Molecular Biology Reports - Male Breast Cancer (MBC) is a rare and aggressive disease that is associated with genetic factors. Mutations in BRCA1 and BRCA2 account for...     

Large scale library generation for high throughput sequencing

Background

Large efforts have recently been made to automate the sample preparation protocols for massively parallel sequencing in order to match the increasing instrument throughput. Still, the size selection through agarose gel electrophoresis separation is a labor-intensive bottleneck of these protocols.

Methodology/Principal Findings

In this study a method for automatic library preparation and size selection on a liquid handling robot is presented. The method utilizes selective precipitation of certain sizes of DNA molecules on to paramagnetic beads for cleanup and selection after standard enzymatic reactions.

Conclusions/Significance

The method is used to generate libraries for de novo and re-sequencing on the Illumina HiSeq 2000 instrument with a throughput of 12 samples per instrument in approximately 4 hours. The resulting output data show quality scores and pass filter rates comparable to manually prepared samples. The sample size distribution can be adjusted for each application, and are suitable for all high throughput DNA processing protocols seeking to control size intervals.     

Evaluation of microbial population dynamics in the co-composting of cow manure and rice straw using high throughput sequencing analysis

Microbial population dynamics in co-composting of cow manure and rice straw were evaluated using 16S high throughput sequencing technology. Physicochemical factors, including temperature, pH, nitrogen contents, the ratio of carbon and nitrogen, and germination index, were also determined in this study. 16S high throughput sequencing results showed that bacterial community structure and composition significantly varied in each phase of composting. The major phyla included Bacteroidetes, Proteobacteria, Firmicutes, Actinobacteria and Planctomycetes, respectively. Bacteroidetes and Proteobacteria were the most abundant phyla in all phases, and Actinobacteria was just dominant in the mesophilic phase, while Firmicutes and Planctomycetes were ubiquitous. At the genus level, Simiduia, Flavobacterium, unclassified Chitinophagaceae and Flexibacter notably changed in each phase of composting. Bacterial community diversity in the mesophilic phase was higher than that in others based on the Shannon–Wiener index and Simpson diversity index. The ratio of carbon and nitrogen and germination index indicated that the co-composting of cow manure and rice straw reached maturation. The result of nitrogen contents showed that nitrogen loss mainly occurred in the thermophilic phase. In addition, the differences in the distributions of key OTUs between in the late thermophilic phase and the cooling and maturation phase were unobvious compared with other phase’s base on the principal component analysis. Redundancy analysis revealed that the changes of nitrogen played a predominant role in the distributions of OTUs during the composting process.     

Rapid genotyping of soybean cultivars using high throughput sequencing

Soybean (Glycine max) breeding involves improving commercially grown varieties by introgressing important agronomic traits from poor yielding accessions and/or wild relatives of soybean while minimizing the associated yield drag. Molecular markers associated with these traits are instrumental in increasing the efficiency of producing such crosses and Single Nucleotide Polymorphisms (SNPs) are particularly well suited for this task, owing to high density in the non-genic regions and thus increased likelihood of finding a tightly linked marker to a given trait. A rapid method to develop SNP markers that can differentiate specific loci between any two parents in soybean is thus highly desirable. In this study we investigate such a protocol for developing SNP markers between multiple soybean accessions and the reference Williams 82 genome. To restrict sampling frequency reduced representation libraries (RRLs) of genomic DNA were generated by restriction digestion followed by library construction. We chose to sequence four accessions Dowling (PI 548663), Dwight (PI 597386), Komata (PI200492) and PI 594538A for their agronomic importance as well as Williams 82 as a control.MseI was chosen to digest genomic DNA based on predictions that it will cut sparingly in the mathematically defined high-copy-number regions of the genome. All RRLs were sequenced on the Illumina genome analyzer. Reads were aligned to the Glyma1 reference assembly and SNP calls made from the alignments. We identified from 4294 to 14550 SNPs between the four accessions and the Williams 82 reference. In addition a small number of SNPs (1142) were found by aligning Williams 82 reads to the reference assembly (Glyma1) suggesting limited genetic variation within the Williams 82 line. The SNP data allowed us to estimate genetic diversity between the four lines and Williams 82. Restriction digestion of soybean genomic DNA with MseI followed by high throughput sequencing provides a rapid and reproducible method for generating SNP markers.     

Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) are endogenous regulators of a broad range of physiological processes and act by either degrading mRNA or blocking its translation. Oilseed rape (Brassica napus) is one of the most important crops in China, Europe and other Asian countries with publicly available expressed sequence tags (ESTs) and genomic survey sequence (GSS) databases, but little is known about its miRNAs and their targets. To date, only 46 miRNAs have been identified in B. napus. RESULTS: Forty-one conserved and 62 brassica-specific candidate B. napus miRNAs, including 20 miRNA* sequences, were identified using Solexa sequencing technology. Furthermore, 33 non-redundant mRNA targets of conserved brassica miRNAs and 19 new non-redundant mRNA targets of novel brassica-specific miRNAs were identified by genome-scale sequencing of mRNA degradome. CONCLUSIONS: This study describes large scale cloning and characterization of B. napus miRNAs and their potential targets, providing the foundation for further characterization of miRNA function in the regulation of diverse physiological processes in B. napus.     

Rethinking microbial diversity analysis in the high throughput sequencing era

The analysis of amplified and sequenced 16S rRNA genes has become the most important single approach for microbial diversity studies. The new sequencing technologies allow for sequencing thousands of reads in a single run and a cost-effective option is split into a single run across many samples. However for this type of investigation the key question that needs to be answered is how many samples can be sequenced without biasing the results due to lack of sequence representativeness? In this work we demonstrated that the level of sequencing effort used for analyzing soil microbial communities biases the results and determines the most effective type of analysis for small and large datasets. Many simulations were performed with four independent pyrosequencing-generated 16S rRNA gene libraries from different environments. The analysis performed here illustrates the lack of resolution of OTU-based approaches for datasets with low sequence coverage. This analysis should be performed with at least 90% of sequence coverage. Diversity index values increase with sample size making normalization of the number of sequences in all samples crucial. An important finding of this study was the advantage of phylogenetic approaches for examining microbial communities with low sequence coverage. However, if the environments being compared were closely related, a deeper sequencing would be necessary to detect the variation in the microbial composition.     

Discovery of transgene insertion sites by high throughput sequencing of mate pair libraries

Background

Transgenesis by random integration of a transgene into the genome of a zygote has become a reliable and powerful method for the creation of new mouse strains that express exogenous genes, including human disease genes, tissue specific reporter genes or genes that allow for tissue specific recombination. Nearly 6,500 transgenic alleles have been created by random integration in embryos over the last 30 years, but for the vast majority of these strains, the transgene insertion sites remain uncharacterized.

Results

To obtain a complete understanding of how insertion sites might contribute to phenotypic outcomes, to more cost effectively manage transgenic strains, and to fully understand mechanisms of instability in transgene expression, we’ve developed methodology and a scoring scheme for transgene insertion site discovery using high throughput sequencing data.

Conclusions

Similar to other molecular approaches to transgene insertion site discovery, high-throughput sequencing of standard paired-end libraries is hindered by low signal to noise ratios. This problem is exacerbated when the transgene consists of sequences that are also present in the host genome. We’ve found that high throughput sequencing data from mate-pair libraries are more informative when compared to data from standard paired end libraries. We also show examples of the genomic regions that harbor transgenes, which have in common a preponderance of repetitive sequences.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-367) contains supplementary material, which is available to authorized users.     

Characterization of bovine miRNAs by sequencing and bioinformatics analysis

Background  

MicroRNAs (miRNAs) are a family of ~22 nucleotide small RNA molecules which regulate gene expression by fully or partially binding to their complementary sequences in mRNAs or promoters. A large number of miRNAs and their expression patterns have been reported in human, mouse and rat. However, miRNAs and their expression patterns in live stock species such as beef cattle are not well studied.     

An efficient, automatable template preparation for high throughput sequencing

We have developed a 96-well format for DNA template isolation that can be readily automatable. The template isolation protocol involves simple alkaline lysis chemistry and reversible capture on a silica solid phase. After the cells are lysed, no centrifugation is necessary, as lysate purification, DNA binding, washing, and release occur in 96-well filter plates. Large numbers of templates prepared using the silica purification method have been sequenced and analyzed. The quality of sequence resulting from our method has been compared with that generated from several commercial plasmid preparation protocols. We found sequence quality of the silica bead preparations to be equivalent to or, in some cases, better than those prepared by other methods. This method offers many advantages over other protocols we have used. First, the silica purifications have allowed us to more than double overall laboratory throughput while decreasing our template isolation materials cost at least five-fold. Second, because we have eliminated all centrifugation steps in the protocol, automation has been much simpler. The protocol has also been adapted to purify PCR products for use as templates in subsequent sequencing reactions.