A transcriptomic analysis of striped catfish (Pangasianodon hypophthalmus) in response to salinity adaptation: De novo assembly, gene annotation and marker discovery

The striped catfish (Pangasianodon hypophthalmus) culture industry in the Mekong Delta in Vietnam has developed rapidly over the past decade. The culture industry now however, faces some significant challenges, especially related to climate change impacts notably from predicted extensive saltwater intrusion into many low topographical coastal provinces across the Mekong Delta. This problem highlights a need for development of culture stocks that can tolerate more saline culture environments as a response to expansion of saline water-intruded land. While a traditional artificial selection program can potentially address this need, understanding the genomic basis of salinity tolerance can assist development of more productive culture lines. The current study applied a transcriptomic approach using Ion PGM technology to generate expressed sequence tag (EST) resources from the intestine and swim bladder from striped catfish reared at a salinity level of 9 ppt which showed best growth performance. Total sequence data generated was 467.8 Mbp, consisting of 4,116,424 reads with an average length of 112 bp. De novo assembly was employed that generated 51,188 contigs, and allowed identification of 16,116 putative genes based on the GenBank non-redundant database. GO annotation, KEGG pathway mapping, and functional annotation of the EST sequences recovered with a wide diversity of biological functions and processes. In addition, more than 11,600 simple sequence repeats were also detected. This is the first comprehensive analysis of a striped catfish transcriptome, and provides a valuable genomic resource for future selective breeding programs and functional or evolutionary studies of genes that influence salinity tolerance in this important culture species.

Assembly and annotation of a non-model gastropod (Nerita melanotragus) transcriptome: a comparison of De novo assemblers

Background The sequencing, de novo assembly and annotation of transcriptome datasets generated with next generation sequencing (NGS) has enabled biologists to answer genomic questions in non-model species with unprecedented ease. Reliable and accurate de novo assembly and annotation of transcriptomes, however, is a critically important step for transcriptome assemblies generated from short read sequences. Typical benchmarks for assembly and annotation reliability have been performed with model species. To address the reliability and accuracy of de novo transcriptome assembly in non-model species, we generated an RNAseq dataset for an intertidal gastropod mollusc species, Nerita melanotragus, and compared the assembly produced by four different de novo transcriptome assemblers; Velvet, Oases, Geneious and Trinity, for a number of quality metrics and redundancy. Results Transcriptome sequencing on the Ion Torrent PGM™ produced 1,883,624 raw reads with a mean length of 133 base pairs (bp). Both the Trinity and Oases de novo assemblers produced the best assemblies based on all quality metrics including fewer contigs, increased N50 and average contig length and contigs of greater length. Overall the BLAST and annotation success of our assemblies was not high with only 15-19% of contigs assigned a putative function. Conclusions We believe that any improvement in annotation success of gastropod species will require more gastropod genome sequences, but in particular an increase in mollusc protein sequences in public databases. Overall, this paper demonstrates that reliable and accurate de novo transcriptome assemblies can be generated from short read sequencers with the right assembly algorithms. Keywords: Nerita melanotragus; De novo assembly; Transcriptome; Heat shock protein; Ion torrent

Optimizing de novo transcriptome assembly and extending genomic resources for striped catfish (Pangasianodon hypophthalmus)

Striped catfish (Pangasianodon hypophthalmus) is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The culture industry is facing a significant challenge however from saltwater intrusion into many low topographical coastal provinces across the Mekong Delta as a result of predicted climate change impacts. Developing genomic resources for this species can facilitate the production of improved culture lines that can withstand raised salinity conditions, and so we have applied high-throughput Ion Torrent sequencing of transcriptome libraries from six target osmoregulatory organs from striped catfish as a genomic resource for use in future selection strategies. We obtained 12,177,770 reads after trimming and processing with an average length of 97 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 66,451 contigs with an average length of 478 bp and N50 length of 506 bp. A total of 37,969 contigs (57%) possessed significant similarity with proteins in the non-redundant database. Comparative analyses revealed that a significant number of contigs matched sequences reported in other teleost fishes, ranging in similarity from 45.2% with Atlantic cod to 52% with zebrafish. In addition, 28,879 simple sequence repeats (SSRs) and 55,721 single nucleotide polymorphisms (SNPs) were detected in the striped catfish transcriptome. The sequence collection generated in the current study represents the most comprehensive genomic resource for P. hypophthalmus available to date. Our results illustrate the utility of next-generation sequencing as an efficient tool for constructing a large genomic database for marker development in non-model species.

De novo combination therapy adefovir plus lamivudine as a treatment for women of child-bearing age with HBeAg-positive chronic hepatitis B before pregnancy

Substantial progress has been achieved in antiviral therapy for chronic hepatitis B; however, options for women of child-bearing age with HBeAg-positive chronic hepatitis B remain a challenge. In this study, we sought to determine whether de novo combination therapy of Adefovir plus Lamivudine was a super treatment for women of child-bearing age with HBeAg-positive chronic hepatitis B prior to conception. A total of 122 women patients of child-bearing age with HBeAg-positive chronic hepatitis B were randomly assigned to receive (i) 10 mg Adefovir plus 100 mg Lamivudine (64 patients) or (ii) 10 mg Adefovir monotherapy (58 patients), administrated orally once daily for 96 weeks. The therapeutic efficacy within each group was compared at weeks 48 and 96. The results showed that de novo combination therapy of Adefovir plus Lamivudine significantly reduced HBV-DNA detectability, and enhanced ALT normalization and HBeAg seroconversion in women of child-bearing age with HBeAg-positive chronic hepatitis B. No virological breakthrough and genotypic resistance were observed in the combination therapy group. Additionally, the combination therapy with Adefovir plus Lamivudine was well tolerated. This study suggests that de novo combination therapy of Adefovir plus Lamivudine offers a therapeutic advantage for women of child-bearing age with HBeAg-positive chronic hepatitis B when taken before conception.

Combining transcriptome assemblies from multiple de novo assemblers in the allo-tetraploid plant Nicotiana benthamiana

Background Nicotiana benthamiana is an allo-tetraploid plant, which can be challenging for de novo transcriptome assemblies due to homeologous and duplicated gene copies. Transcripts generated from such genes can be distinct yet highly similar in sequence, with markedly differing expression levels. This can lead to unassembled, partially assembled or mis-assembled contigs. Due to the different properties of de novo assemblers, no one assembler with any one given parameter space can re-assemble all possible transcripts from a transcriptome. Results In an effort to maximise the diversity and completeness of de novo assembled transcripts, we utilised four de novo transcriptome assemblers, TransAbyss, Trinity, SOAPdenovo-Trans, and Oases, using a range of k-mer sizes and different input RNA-seq read counts. We complemented the parameter space biologically by using RNA from 10 plant tissues. We then combined the output of all assemblies into a large super-set of sequences. Using a method from the EvidentialGene pipeline, the combined assembly was reduced from 9.9 million de novo assembled transcripts to about 235,000 of which about 50,000 were classified as primary. Metrics such as average bit-scores, feature response curves and the ability to distinguish paralogous or homeologous transcripts, indicated that the EvidentialGene processed assembly was of high quality. Of 35 RNA silencing gene transcripts, 34 were identified as assembled to full length, whereas in a previous assembly using only one assembler, 9 of these were partially assembled. Conclusions To achieve a high quality transcriptome, it is advantageous to implement and combine the output from as many different de novo assemblers as possible. We have in essence taking the ‘best’ output from each assembler while minimising sequence redundancy. We have also shown that simultaneous assessment of a variety of metrics, not just focused on contig length, is necessary to gauge the quality of assemblies.

A soluble preparation from developing groundnut seeds (Arachis hypogaea) catalyzes de novo synthesis of long chain fatty acids

A 100,000 x g supernatant fraction prepared from developing groundnut seeds (30-35 days after flowering) catalyzed the synthesis of fatty acids from [l-14C]acetate at a rate of 120nmoles of acetate incorporated per hr per gram fresh weight of tissue. 90% of this incorporated label was associated with fatty acids. The major fatty acids formed were stearic- (77%) and palmitic acids (14%) with 4% of oleic acid. The fatty acid synthetase activity was stable when stored at 0-4 degrees C for at least fifteen days. It is concluded from these results that acetyl-coA carboxylase and all the enzymes of fatty acid synthetase from developing groundnut seeds are soluble.

A transcriptomic analysis of the kidney tissue of Tra catfish (pangasianodon hypophthalmus) reared in saline condition: De novo assembly, annotation, SNP discovery

Pangasianodon hypophthalmus is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The current study using Ion Torrent technology generated EST resources from the kidney for Tra catfish reared at a salinity level of 9 ppt. We obtained 2,623,929 reads after trimming and processing with an average length of 104 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 29,940 contigs, and allowing identification of 5,710 putative genes when comppared with NCBI non-redundant database. A large number of single nucleotide polymorphisms (SNPs) were also detected. The sequence collection generated in our study represents the most comprehensive transcriptomic resource for P. hypophthalmus available to date.

Map of the Novo-Shvedsʻka colony

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De novo synthesis of acetylcholinesterase in roots of Pisum sativum

Pisum sativum seeds contain a conserved acetylcholinesterase (AChE) which is active during the early stages of germination. The enzyme activity soon disappears and reappears after 72 hr of germination. A protein devoid of catalytic ability, but exhibiting similar chromatographic and electrophoretic properties as the active AChE, could be detected after 24 hr of germination. The pattern of incorporation of labelled amino acids into AChE and the influence of cycloheximide revealed that the AChE found in the roots from 72 hr onwards was entirely new. During this period of growth, the AChE protein accounts for 4–10% of the total proteins in the root tissue.

De novo synthesis of 1-deoxythiosugars

A short and efficient chemical synthesis of biologically potent and novel 1-deoxythiosugars is accomplished. Introduction of sulfur mediated by benzyltriethylammonium tetrathiomolybdate, as a sulfur transfer reagent through nucleophilic double displacement of tosylate in alpha,omega-di-O-tosyl aldonolactones in an intramolecular fashion is the key feature. The subsequent reduction of thiosugar lactones with borohydride exchange resin (BER) offers a number of deoxythiosugars in good overall yield. (C) 2013 Elsevier Ltd. All rights reserved.

De novo inference of protein function from coarse-grained dynamics

Inference of molecular function of proteins is the fundamental task in the quest for understanding cellular processes. The task is getting increasingly difficult with thousands of new proteins discovered each day. The difficulty arises primarily due to lack of high-throughput experimental technique for assessing protein molecular function, a lacunae that computational approaches are trying hard to fill. The latter too faces a major bottleneck in absence of clear evidence based on evolutionary information. Here we propose a de novo approach to annotate protein molecular function through structural dynamics match for a pair of segments from two dissimilar proteins, which may share even <10% sequence identity. To screen these matches, corresponding 1 mu s coarse-grained (CG) molecular dynamics trajectories were used to compute normalized root-mean-square-fluctuation graphs and select mobile segments, which were, thereafter, matched for all pairs using unweighted three-dimensional autocorrelation vectors. Our in-house custom-built forcefield (FF), extensively validated against dynamics information obtained from experimental nuclear magnetic resonance data, was specifically used to generate the CG dynamics trajectories. The test for correspondence of dynamics-signature of protein segments and function revealed 87% true positive rate and 93.5% true negative rate, on a dataset of 60 experimentally validated proteins, including moonlighting proteins and those with novel functional motifs. A random test against 315 unique fold/function proteins for a negative test gave >99% true recall. A blind prediction on a novel protein appears consistent with additional evidences retrieved therein. This is the first proof-of-principle of generalized use of structural dynamics for inferring protein molecular function leveraging our custom-made CG FF, useful to all. (C) 2014 Wiley Periodicals, Inc.