A Case-Only Genome-wide Association Study of Gender- and Age-specific Risk Markers for Childhood Leukemia

Males and age group 1 to 5 years show a much higher risk for childhood acute lymphoblastic leukemia (ALL). We performed a case-only genome-wide association study (GWAS), using the Illumina Infinium HumanCoreExome Chip, to unmask gender- and age-specific risk variants in 240 non-Hispanic white children with ALL recruited at Texas Children’s Cancer Center, Houston, Texas. Besides statistically most significant results, we also considered results that yielded the highest effect sizes. Existing experimental data and bioinformatic predictions were used to complement results, and to examine the biological significance of statistical results. Our study identified novel risk variants for childhood ALL. The SNP, rs4813720 (RASSF2), showed the statistically most significant gender-specific associations (P < 2 x 10-6). Likewise, rs10505918 (SOX5) yielded the lowest P value (P < 1 x 10-5) for age-specific associations, and also showed the statistically most significant association with age-at-onset (P < 1 x 10-4). Two SNPs, rs12722042 and 12722039, from the HLA-DQA1 region yielded the highest effect sizes (odds ratio (OR) = 15.7; P = 0.002) for gender-specific results, and the SNP, rs17109582 (OR = 12.5; P = 0.006), showed the highest effect size for age-specific results. Sex chromosome variants did not appear to be involved in gender-specific associations. The HLA-DQA1 SNPs belong to DQA1*01:07and confirmed previously reported male-specific association with DQA1*01:07. Twenty one of the SNPs identified as risk markers for gender- or age-specific associations were located in the transcription factor binding sites and 56 SNPs were non-synonymous variants, likely to alter protein function. Although bioinformatic analysis did not implicate a particular mechanism for gender- and age-specific associations, RASSF2 has an estrogen receptor-alpha binding site in its promoter. The unknown mechanisms may be due to lack of interest in gender- and age-specificity in associations. These results provide a foundation for further studies to examine the gender- and age-differential in childhood ALL risk. Following replication and mechanistic studies, risk factors for one gender or age group may have a potential to be used as biomarkers for targeted intervention for prevention and maybe also for treatment.

Sequence and Structural Determinants of Specificity Differences between Paralogous Transcription Factors

Transcription factors (TFs) control the temporal and spatial expression of target genes by interacting with DNA in a sequence-specific manner. Recent advances in high throughput experiments that measure TF-DNA interactions in vitro and in vivo have facilitated the identification of DNA binding sites for thousands of TFs. However, it remains unclear how each individual TF achieves its specificity, especially in the case of paralogous TFs that recognize distinct target genomic sites despite sharing very similar DNA binding motifs. In my work, I used a combination of high throughput in vitro protein-DNA binding assays and machine-learning algorithms to characterize and model the binding specificity of 11 paralogous TFs from 4 distinct structural families. My work proves that even very closely related paralogous TFs, with indistinguishable DNA binding motifs, oftentimes exhibit differential binding specificity for their genomic target sites, especially for sites with moderate binding affinity. Importantly, the differences I identify in vitro and through computational modeling help explain, at least in part, the differential in vivo genomic targeting by paralogous TFs. Future work will focus on in vivo factors that might also be important for specificity differences between paralogous TFs, such as DNA methylation, interactions with protein cofactors, or the chromatin environment. In this larger context, my work emphasizes the importance of intrinsic DNA binding specificity in targeting of paralogous TFs to the genome.

Genome analysis and characterisation of the exopolysaccharide produced by Bifidobacterium longum subsp. longum 35624™

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPS624). Comparative genome analysis of the 35624 strain with other B. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPS624. Following isolation and acid treatment of the EPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide.

Draft Genome Sequence of Staphylococcus succinus Strain CSM-77, a Moderately Halophilic Bacterium Isolated from a Triassic Salt Mine.

Here, we report the draft genome sequence of Staphylococcus succinus strain CSM-77. This moderately halophilic bacterium was isolated from the surface of a halite sample obtained from a Triassic salt mine.

First complete genome sequence of a capsicum chlorosis tospovirus isolate from Australia with an unusually large S RNA intergenic region

The first complete genome sequence of capsicum chlorosis virus (CaCV) from Australia was determined using a combination of Illumina HiSeq RNA and Sanger sequencing technologies. Australian CaCV had a tripartite genome structure like other CaCV isolates. The large (L) RNA was 8913 nucleotides (nt) in length and contained a single open reading frame (ORF) of 8634 nt encoding a predicted RNA-dependent RNA polymerase (RdRp) in the viral-complementary (vc) sense. The medium (M) and small (S) RNA segments were 4846 and 3944 nt in length, respectively, each containing two non-overlapping ORFs in ambisense orientation, separated by intergenic regions (IGR). The M segment contained ORFs encoding the predicted non-structural movement protein (NSm; 927 nt) and precursor of glycoproteins (GP; 3366 nt) in the viral sense (v) and vc strand, respectively, separated by a 449-nt IGR. The S segment coded for the predicted nucleocapsid (N) protein (828 nt) and non-structural suppressor of silencing protein (NSs; 1320 nt) in the vc and v strand, respectively. The S RNA contained an IGR of 1663 nt, being the largest IGR of all CaCV isolates sequenced so far. Comparison of the Australian CaCV genome with complete CaCV genome sequences from other geographic regions showed highest sequence identity with a Taiwanese isolate. Genome sequence comparisons and phylogeny of all available CaCV isolates provided evidence for at least two highly diverged groups of CaCV isolates that may warrant re-classification of AIT-Thailand and CP-China isolates as unique tospoviruses, separate from CaCV.

Whole Genome Analysis of Gene Expression Reveals Coordinated Activation of Signaling and Metabolic Pathways during Pollen-Pistil Interactions in Arabidopsis

Plant reproduction depends on the concerted activation of many genes to ensure correct communication between pollen and pistil. Here, we queried the whole transcriptome of Arabidopsis (Arabidopsis thaliana) in order to identify genes with specific reproductive functions. We used the Affymetrix ATH1 whole genome array to profile wild-type unpollinated pistils and unfertilized ovules. By comparing the expression profile of pistils at 0.5, 3.5, and 8.0 h after pollination and applying a number of statistical and bioinformatics criteria, we found 1,373 genes differentially regulated during pollen-pistil interactions. Robust clustering analysis grouped these genes in 16 time-course clusters representing distinct patterns of regulation. Coregulation within each cluster suggests the presence of distinct genetic pathways, which might be under the control of specific transcriptional regulators. A total of 78% of the regulated genes were expressed initially in unpollinated pistil and/or ovules, 15% were initially detected in the pollen data sets as enriched or preferentially expressed, and 7% were induced upon pollination. Among those, we found a particular enrichment for unknown transcripts predicted to encode secreted proteins or representing signaling and cell wall-related proteins, which may function by remodeling the extracellular matrix or as extracellular signaling molecules. A strict regulatory control in various metabolic pathways suggests that fine-tuning of the biochemical and physiological cellular environment is crucial for reproductive success. Our study provides a unique and detailed temporal and spatial gene expression profile of in vivo pollen-pistil interactions, providing a framework to better understand the basis of the molecular mechanisms operating during the reproductive process in higher plants.

Near complete genome sequence of the animal feed probiotic, Bacillus amyloliquefaciens H57

Bacillus amyloliquefaciens H57 is a bacterium isolated from lucerne for its ability to prevent feed spoilage. Further interest developed when ruminants fed with H57-inoculated hay showed increased weight gain and nitrogen retention relative to controls, suggesting a probiotic effect. The near complete genome of H57 is ~3.96 Mb comprising 16 contigs. Within the genome there are 3,836 protein coding genes, an estimated sixteen rRNA genes and 69 tRNA genes. H57 has the potential to synthesise four different lipopeptides and four polyketide compounds, which are known antimicrobials. This antimicrobial capacity may facilitate the observed probiotic effect.

Evolution of genome sequencing techniques

The quality and the speed for genome sequencing has advanced at the same time that technology boundaries are stretched. This advancement has been divided so far in three generations. The first-generation methods enabled sequencing of clonal DNA populations. The second-generation massively increased throughput by parallelizing many reactions while the third-generation methods allow direct sequencing of single DNA molecules. The first techniques to sequence DNA were not developed until the mid-1970s, when two distinct sequencing methods were developed almost simultaneously, one by Alan Maxam and Walter Gilbert, and the other one by Frederick Sanger. The first one is a chemical method to cleave DNA at specific points and the second one uses ddNTPs, which synthesizes a copy from the DNA chain template. Nevertheless, both methods generate fragments of varying lengths that are further electrophoresed. Moreover, it is important to say that until the 1990s, the sequencing of DNA was relatively expensive and it was seen as a long process. Besides, using radiolabeled nucleotides also compounded the problem through safety concerns and prevented the automation. Some advancements within the first generation include the replacement of radioactive labels by fluorescent labeled ddNTPs and cycle sequencing with thermostable DNA polymerase, which allows automation and signal amplification, making the process cheaper, safer and faster. Another method is Pyrosequencing, which is based on the “sequencing by synthesis” principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation. By the end of the last millennia, parallelization of this method started the Next Generation Sequencing (NGS) with 454 as the first of many methods that can process multiple samples, calling it the 2º generation sequencing. Here electrophoresis was completely eliminated. One of the methods that is sometimes used is SOLiD, based on sequencing by ligation of fluorescently dye-labeled di-base probes which competes to ligate to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction. The widely used Solexa/Illumina method uses modified dNTPs containing so called “reversible terminators” which blocks further polymerization. The terminator also contains a fluorescent label, which can be detected by a camera. Now, the previous step towards the third generation was in charge of Ion Torrent, who developed a technique that is based in a method of “sequencing-by-synthesis”. Its main feature is the detection of hydrogen ions that are released during base incorporation. Likewise, the third generation takes into account nanotechnology advancements for the processing of unique DNA molecules to a real time synthesis sequencing system like PacBio; and finally, the NANOPORE, projected since 1995, also uses Nano-sensors forming channels obtained from bacteria that conducts the sample to a sensor that allows the detection of each nucleotide residue in the DNA strand. The advancements in terms of technology that we have nowadays have been so quick, that it makes wonder: ¿How do we imagine the next generation?

Complete Genome Sequence of the Hyperthermophilic and Piezophilic Archeon Thermococcus piezophilus CDGS T , Able To Grow under Extreme Hydrostatic Pressures

We report the genome sequence of Thermococcus superprofundus strain CDGST, a new piezophilic and hyperthermophilic member of the order Thermococcales isolated from the world’s deepest hydrothermal vents, at the Mid-Cayman Rise. The genome is consistent with a heterotrophic, anaerobic, and piezophilic lifestyle.

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous, multidrug resistant, biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniae species from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1, blaSHV-28, aac(6’)1b-cr, catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1, aac(6’)-Ib, aac(3)-IId, sul1,2, blaTEM-1A,1B, blaOXA-9, blaCTX-M-15, blaSHV-11, cmlA1, erm(B), mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniae strains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniae NTUHK2044, a transposase gene InsH of IS5-13 was found inserted.

Complete genome sequence of Deltapapillomavirus 4 (bovine papillomavirus 2) from a bovine papillomavirus lesion in Amazon Region, Brazil

The complete genome sequence of bovine papillomavirus 2 (BPV2) from Brazilian Amazon Region was determined using multiple-primed rolling circle amplification followed by Illumina sequencing. The genome is 7,947 bp long, with 45.9% GC content. It encodes seven early (E1, E2, E4, E5, E6, E7, and E8) and two late (L1 and L2) genes. The complete genome of a BPV2 can help in future studies since this BPV type is highly reported worldwide although the lack of complete genome sequences available.

A case-only genome-wide association study of gender- and age-specific risk markers for childhood leukemia

Males and age group 1 to 5 years show a much higher risk for childhood acute lymphoblastic leukemia (ALL). We performed a case-only genome-wide association study (GWAS), using the Illumina Infinium HumanCoreExome Chip, to unmask gender- and age-specific risk variants in 240 non-Hispanic white children with ALL recruited at Texas Children’s Cancer Center, Houston, Texas. Besides statistically most significant results, we also considered results that yielded the highest effect sizes. Existing experimental data and bioinformatic predictions were used to complement results, and to examine the biological significance of statistical results. ^ Our study identified novel risk variants for childhood ALL. The SNP, rs4813720 (RASSF2), showed the statistically most significant gender-specific associations (P < 2 x 10-6). Likewise, rs10505918 (SOX5) yielded the lowest P value (P < 1 x 10-5 ) for age-specific associations, and also showed the statistically most significant association with age-at-onset (P < 1 x 10-4). Two SNPs, rs12722042 and 12722039, from the HLA-DQA1 region yielded the highest effect sizes (odds ratio (OR) = 15.7; P = 0.002) for gender-specific results, and the SNP, rs17109582 (OR = 12.5; P = 0.006), showed the highest effect size for age-specific results. Sex chromosome variants did not appear to be involved in gender-specific associations. ^ The HLA-DQA1 SNPs belong to DQA1*01:07and confirmed previously reported male-specific association with DQA1*01:07. Twenty one of the SNPs identified as risk markers for gender- or age-specific associations were located in the transcription factor binding sites and 56 SNPs were non-synonymous variants, likely to alter protein function. Although bioinformatic analysis did not implicate a particular mechanism for gender- and age-specific associations, RASSF2 has an estrogen receptor-alpha binding site in its promoter. The unknown mechanisms may be due to lack of interest in gender- and age-specificity in associations. These results provide a foundation for further studies to examine the gender- and age-differential in childhood ALL risk. Following replication and mechanistic studies, risk factors for one gender or age group may have a potential to be used as biomarkers for targeted intervention for prevention and maybe also for treatment.^

Genome sequence of Lactococcus garvieae 8831, isolated from rainbow trout lactococcosis outbreaks in Spain

Lactococcus garvieae is the etiological agent of lactococcosis, one of the most important disease threats to the sustainability of the rainbow trout farming industry. Here, we present the draft genome sequence of Lactococcus garvieae strain 8831, isolated from diseased rainbow trout, which is composed of 2,087,276 bp with a G+C content of 38%.

Genome sequence of Lactococcus garvieae 21881, isolated in a case of human septicemia

Lactococcus garvieae is a Gram-positive bacterium considered an important opportunistic emerging human pathogen and also a well-recognized fish pathogen. Here, we present the draft genome sequence of Lactococcus garvieae strain 21881 (2,164,557 bp, with a G+C content of 37.9%), which represents the first report of a genome sequence on Lactococcus garvieae.