Aldrin Kay-Yuen Yim

Identification of a novel salt tolerance gene in wild soybean by whole-genome sequencing

Using a whole-genome-sequencing approach to explore germplasm resources can serve as an important strategy for crop improvement, especially in investigating wild accessions that may contain useful genetic resources that have been lost during the domestication process. Here we sequence and assemble a draft genome of wild soybean and construct a recombinant inbred population for genotyping-by-sequencing and phenotypic analyses to identify multiple QTLs relevant to traits of interest in agriculture. We use a combination of de novo sequencing data from this work and our previous germplasm re-sequencing data to identify a novel ion transporter gene, GmCHX1, and relate its sequence alterations to salt tolerance. Rapid gain-of-function tests show the protective effects of GmCHX1 towards salt stress. This combination of whole-genome de novo sequencing, high-density-marker QTL mapping by re-sequencing and functional analyses can serve as an effective strategy to unveil novel genomic information in wild soybean to facilitate crop improvement.

Genome-Wide Structural Variation Detection by Genome Mapping on Nanochannel Arrays.

Comprehensive whole-genome structural variation detection is challenging with current approaches. With diploid cells as DNA source and the presence of numerous repetitive elements, short-read DNA sequencing cannot be used to detect structural variation efficiently. In this report, we show that genome mapping with long, fluorescently labeled DNA molecules imaged on nanochannel arrays can be used for whole-genome structural variation detection without sequencing. While whole-genome haplotyping is not achieved, local phasing (across >150-kb regions) is routine, as molecules from the parental chromosomes are examined separately. In one experiment, we generated genome maps from a trio from the 1000 Genomes Project, compared the maps against that derived from the reference human genome, and identified structural variations that are >5 kb in size. We find that these individuals have many more structural variants than those published, including some with the potential of disrupting gene function or regulation.

Using RNA-Seq Data to Evaluate Reference Genes Suitable for Gene Expression Studies in Soybean

Differential gene expression profiles often provide important clues for gene functions. While reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an important tool, the validity of the results depends heavily on the choice of proper reference genes. In this study, we employed new and published RNA-sequencing (RNA-Seq) datasets (26 sequencing libraries in total) to evaluate reference genes reported in previous soybean studies. In silico PCR showed that 13 out of 37 previously reported primer sets have multiple targets, and 4 of them have amplicons with different sizes. Using a probabilistic approach, we identified new and improved candidate reference genes. We further performed 2 validation tests (with 26 RNA samples) on 8 commonly used reference genes and 7 newly identified candidates, using RT-qPCR. In general, the new candidate reference genes exhibited more stable expression levels under the tested experimental conditions. The three newly identified candidate reference genes Bic-C2, F-box protein2, and VPS-like gave the best overall performance, together with the commonly used ELF1b. It is expected that the proposed probabilistic model could serve as an important tool to identify stable reference genes when more soybean RNA-Seq data from different growth stages and treatments are used.

Mutations Enabling Displacement of Tryptophan by 4-Fluorotryptophan as a Canonical Amino Acid of the Genetic Code

The 20 canonical amino acids of the genetic code have been invariant over 3 billion years of biological evolution. Although various aminoacyl-tRNA synthetases can charge their cognate tRNAs with amino acid analogs, there has been no known displacement of any canonical amino acid from the code. Experimental departure from this universal protein alphabet comprising the canonical amino acids was first achieved in the mutants of the Bacillus subtilis QB928 strain, which after serial selection and mutagenesis led to the HR23 strain that could use 4-fluorotryptophan (4FTrp) but not canonical tryptophan (Trp) for propagation. To gain insight into this displacement of Trp from the genetic code by 4FTrp, genome sequencing was performed on LC33 (a precursor strain of HR23), HR23, and TR7 (a revertant of HR23 that regained the capacity to propagate on Trp). Compared with QB928, the negative regulator mtrB of Trp transport was found to be knocked out in LC33, HR23, and TR7, and sigma factor sigB was mutated in HR23 and TR7. Moreover, rpoBC encoding RNA polymerase subunits were mutated in three independent isolates of TR7 relative to HR23. Increased expression of sigB was also observed in HR23 and in TR7 growing under 4FTrp. These findings indicated that stabilization of the genetic code can be provided by just a small number of analog-sensitive proteins, forming an oligogenic barrier that safeguards the canonical amino acids throughout biological evolution.

Organism-Specific rRNA Capture System for Application in Next-Generation Sequencing

RNA-sequencing is a powerful tool in studying RNomics. However, the highly abundance of ribosomal RNAs (rRNA) and transfer RNA (tRNA) have predominated in the sequencing reads, thereby hindering the study of lowly expressed genes. Therefore, rRNA depletion prior to sequencing is often performed in order to preserve the subtle alteration in gene expression especially those at relatively low expression levels. One of the commercially available methods is to use DNA or RNA probes to hybridize to the target RNAs. However, there is always a concern with the non-specific binding and unintended removal of messenger RNA (mRNA) when the same set of probes is applied to different organisms. The degree of such unintended mRNA removal varies among organisms due to organism-specific genomic variation. We developed a computer-based method to design probes to deplete rRNA in an organism-specific manner. Based on the computation results, biotinylated-RNA-probes were produced by in vitro transcription and were used to perform rRNA depletion with subtractive hybridization. We demonstrated that the designed probes of 16S rRNAs and 23S rRNAs can efficiently remove rRNAs from Mycobacterium smegmatis. In comparison with a commercial subtractive hybridization-based rRNA removal kit, using organism-specific probes is better in preserving the RNA integrity and abundance. We believe the computer-based design approach can be used as a generic method in preparing RNA of any organisms for next-generation sequencing, particularly for the transcriptome analysis of microbes.

The Essential Component in DNA-Based Information Storage System: Robust Error-Tolerating Module.

The size of digital data is ever increasing and is expected to grow to 40,000 EB by 2020, yet the estimated global information storage capacity in 2011 is <300 EB, indicating that most of the data are transient. DNA, as a very stable nano-molecule, is an ideal massive storage device for long-term data archive. The two most notable illustrations are from Church et al. and Goldman et al., whose approaches are well-optimized for most sequencing platforms – short synthesized DNA fragments without homopolymer. Here, we suggested improvements on error handling methodology that could enable the integration of DNA-based computational process, e.g., algorithms based on self-assembly of DNA. As a proof of concept, a picture of size 438 bytes was encoded to DNA with low-density parity-check error-correction code. We salvaged a significant portion of sequencing reads with mutations generated during DNA synthesis and sequencing and successfully reconstructed the entire picture. A modular-based programing framework – DNAcodec with an eXtensible Markup Language-based data format was also introduced. Our experiments demonstrated the practicability of long DNA message recovery with high error tolerance, which opens the field to biocomputing and synthetic biology.

Detection of splicing events and multiread locations from RNA-seq data based on a geometric-tail (GT) distribution of intron length

BackgroundRNA sequencing (RNA-seq) measures gene expression levels and permits splicing analysis. Many existing aligners are capable of mapping millions of sequencing reads onto a reference genome. For reads that can be mapped to multiple positions along the reference genome (multireads), these aligners may either randomly assign them to a location, or discard them altogether. Either way could bias downstream analyses. Meanwhile, challenges remain in the alignment of reads spanning across splice junctions. Existing splicing-aware aligners that rely on the read-count method in identifying junction sites are inevitably affected by sequencing depths.ResultsThe distance between aligned positions of paired-end (PE) reads or two parts of a spliced read is dependent on the experiment protocol and gene structures. We here proposed a new method that employs an empirical geometric-tail (GT) distribution of intron lengths to make a rational choice in multireads selection and splice-sites detection, according to the aligned distances from PE and sliced reads.ConclusionsGT models that combine sequence similarity from alignment, and together with the probability of length distribution, could accurately determine the location of both multireads and spliced reads.

Comparative transcriptomics of multidrug-resistant Acinetobacter baumannii in response to antibiotic treatments

Multidrug-resistant Acinetobacter baumannii, a major hospital-acquired pathogen, is a serious health threat and poses a great challenge to healthcare providers. Although there have been many genomic studies on the evolution and antibiotic resistance of this species, there have been very limited transcriptome studies on its responses to antibiotics. We conducted a comparative transcriptomic study on 12 strains with different growth rates and antibiotic resistance profiles, including 3 fast-growing pan-drug-resistant strains, under separate treatment with 3 antibiotics, namely amikacin, imipenem, and meropenem. We performed deep sequencing using a strand-specific RNA-sequencing protocol, and used de novo transcriptome assembly to analyze gene expression in the form of polycistronic transcripts. Our results indicated that genes associated with transposable elements generally showed higher levels of expression under antibiotic-treated conditions, and many of these transposon-associated genes have previously been linked to drug resistance. Using co-expressed transposon genes as markers, we further identified and experimentally validated two novel genes of which overexpression conferred significant increases in amikacin resistance. To the best of our knowledge, this study represents the first comparative transcriptomic analysis of multidrug-resistant A. baumannii under different antibiotic treatments, and revealed a new relationship between transposons and antibiotic resistance.

Sexual dimorphism in glioma glycolysis underlies sex differences in survival

The molecular bases for sex differences in cancer remain undefined and how to incorporate them into risk stratification remains undetermined. Given sex differences in metabolism and the inverse correlation between fluorodeoxyglucose (FDG) uptake and survival, we hypothesized that glycolytic phenotyping would improve glioma subtyping. Using retrospectively acquired lower-grade glioma (LGG) transcriptome data from The Cancer Genome Atlas (TCGA), we discovered male-specific decreased survival resulting from glycolytic gene overexpression. Patients within this high-glycolytic group showed significant differences in the presence of key genomic alterations (i.e., 1p/19q codeletion, CIC, EGFR, NF1, PTEN, FUBP1, and IDH mutations) compared with the low-glycolytic group. Although glycolytic stratification defined poor prognostic males independent of grade, histology, TP53, and ATRX mutation status, we unexpectedly found that females with high-glycolytic gene expression and wild-type IDH survived longer than all other wild-type patients. Validation with an independent metabolomics dataset from grade 2 gliomas determined that glycolytic metabolites selectively stratified males and also uncovered a potential sexual dimorphism in pyruvate metabolism. These findings identify a potential synergy between patient sex, tumor metabolism, and genomic alterations in determining outcome for glioma patients.

Draft Genome Sequence of Extensively Drug-Resistant Acinetobacter baumannii Strain CUAB1 from a Patient in Hong Kong, China

ABSTRACT We report the draft genome sequence of an extensively drug-resistant strain of Acinetobacter baumannii, CUAB1, isolated from a patient in a local Hong Kong hospital. MIC testing was performed, and genes previously associated with drug resistance were located.