Variant Identification in Multi-Sample Pools by Illumina Genome Analyzer Sequencing

Multi-sample pooling and Illumina Genome Analyzer (GA) sequencing allows high throughput sequencing of multiple samples to determine population sequence variation. A preliminary experiment, using the RET proto-oncogene as a model, predicted ≤30 samples could be pooled to reliably detect singleton variants without requiring additional confirmation testing. This report used 30 and 50 sample pools to test the hypothesized pooling limit and also to test recent protocol improvements, Illumina GAIIx upgrades, and longer read chemistry. The SequalPrep^TM method was used to normalize amplicons before pooling. For comparison, a single ‘control’ sample was run in a different flow cell lane. Data was evaluated by variant read percentages and the subtractive correction method which utilizes the control sample. In total, 59 variants were detected within the pooled samples, which included all 47 known true variants. The 15 known singleton variants due to Sanger sequencing had an average of 1.62±0.26% variant reads for the 30 pool (expected 1.67% for a singleton variant [unique variant within the pool]) and 1.01±0.19% for the 50 pool (expected 1%). The 76 base read lengths had higher error rates than shorter read lengths (33 and 50 base reads), which eliminated the distinction of true singleton variants from background error. This report demonstrated pooling limits from 30 up to 50 samples (depending on error rates and coverage), for reliable singleton variant detection. The presented pooling protocols and analysis methods can be used for variant discovery in other genes, facilitating molecular diagnostic test design and interpretation.     

Genomic evidences for the existence of a phenylpropanoid metabolic pathway in Aspergillus oryzae

Plants interact with their environment by producing a diverse array of secondary metabolites. A majority of these compounds are phenylpropanoids and flavonoids which are valued for their medicinal and agricultural properties. The phenylpropanoid biosynthesis pathway proceeds with the basic C6-C3 carbon skeleton of phenylalanine, and involves a wide range of enzymes viz., phenylalanine ammonia lyase, coumarate hydroxylase, coumarate ligase, chalcone synthase, chalcone reductase and chalcone isomerase. Recently, bacteria have also been shown to contain homodimeric polyketide synthases belonging to the plant chalcone synthase superfamily linking the capabilities of plants and bacteria in the biosynthesis of flavonoids. We report here the presence of genes encoding the core enzymes of the phenylpropanoid pathway in an industrially useful fungus, Aspergillus oryzae. Although the assignment of enzyme function must be confirmed by further biochemical evidences, this work has allowed us to anticipate the phenylpropanoid metabolism profile in a filamentous fungus for the first time and paves way for research on identifying novel fungal flavonoid-like metabolites.     

Anthocyanin biosynthetic genes in Brassica rapa

Background

Anthocyanins are a group of flavonoid compounds. As a group of important secondary metabolites, they perform several key biological functions in plants. Anthocyanins also play beneficial health roles as potentially protective factors against cancer and heart disease. To elucidate the anthocyanin biosynthetic pathway in Brassica rapa, we conducted comparative genomic analyses between Arabidopsis thaliana and B. rapa on a genome-wide level.

Results

In total, we identified 73 genes in B. rapa as orthologs of 41 anthocyanin biosynthetic genes in A. thaliana. In B. rapa, the anthocyanin biosynthetic genes (ABGs) have expanded and most genes exist in more than one copy. The anthocyanin biosynthetic structural genes have expanded through whole genome and tandem duplication in B. rapa. More structural genes located upstream of the anthocyanin biosynthetic pathway have been retained than downstream. More negative regulatory genes are retained in the anthocyanin biosynthesis regulatory system of B. rapa.

Conclusions

These results will promote an understanding of the genetic mechanism of anthocyanin biosynthesis, as well as help the improvement of the nutritional quality of B. rapa through the breeding of high anthocyanin content varieties.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-426) contains supplementary material, which is available to authorized users.     

Multi-Sample Pooling and Illumina Genome Analyzer Sequencing Methods to Determine Gene Sequence Variation for Database Development

Determination of sequence variation within a genetic locus to develop clinically relevant databases is critical for molecular assay design and clinical test interpretation, so multisample pooling for Illumina genome analyzer (GA) sequencing was investigated using the RET proto-oncogene as a model. Samples were Sanger-sequenced for RET exons 10, 11, and 13–16. Ten samples with 13 known unique variants (“singleton variants” within the pool) and seven common changes were amplified and then equimolar-pooled before sequencing on a single flow cell lane, generating 36 base reads. For comparison, a single “control” sample was run in a different lane. After alignment, a 24-base quality score-screening threshold and 3` read end trimming of three bases yielded low background error rates with a 27% decrease in aligned read coverage. Sequencing data were evaluated using an established variant detection method (percent variant reads), by the presented subtractive correction method, and with SNPSeeker software. In total, 41 variants (of which 23 were singleton variants) were detected in the 10 pool data, which included all Sanger-identified variants. The 23 singleton variants were detected near the expected 5% allele frequency (average 5.17%±0.90% variant reads), well above the highest background error (1.25%). Based on background error rates, read coverage, simulated 30, 40, and 50 sample pool data, expected singleton allele frequencies within pools, and variant detection methods; ≥30 samples (which demonstrated a minimum 1% variant reads for singletons) could be pooled to reliably detect singleton variants by GA sequencing.     

Anti-fungal effects of phenolic amides isolated from the root bark of Lycium chinense

Four phenolic amides, dihydro-N-caffeoyltyramine (1), trans-N-feruloyloctopamine (2), trans-N -caffeoyltyramine (3), and cis-N-caffeoyltyramine (4), were isolated from an ethyl acetate extract of the root bark of Lycium chinense Miller. All had an anti-fungal effect; compounds 1-3 were potent at 5-10 microg ml(-1) and were without hemolytic activity against human erythrocyte cells. Compound 4 was active at 40 microg ml(-1). All four compounds impeded the dimorphic transition of pathogen, Candida albicans.     

Fermentation of a milk-soymilk and Lycium chinense Miller mixture using a new isolate of Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum

A milk–soymilk mixture was fermented using Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum BCRC11847 at different inoculum ratios (1:1, 1:2, 1:5, 2:1, and 5:1). When the inoculum ratio was 1:2, the cell numbers of both strains were balanced after 12 h of cultivation. The pH and titratable acidity were very similar at the various inoculum ratios of cultivation. The milk–soymilk mixture was supplemented with 5, 10, 15, and 20% Lycium chinense Miller juice and fermented with Lactobacillus paracasei subsp. paracasei NTU101 and B. longum BCRC11847. Sensory evaluation results showed that supplementation with 5% Lycium chinense Miller juice improved the acceptability of the fermented milk–soymilk. The fermented beverage was stored at 4°C for 14 days; variations in pH and titratable acidity were slight. The cell numbers of L. paracasei subsp. paracasei NTU101 and B. longum BCRC11847 in the fermented beverage were maintained at 1.2×10⁹ CFU/ml and 6.3×10⁸ CFU/ml, respectively, after 14 days of storage.     

Carotenoid biosynthetic genes in Brassica rapa: comparative genomic analysis,phylogenetic analysis,and expression profiling

Background

Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms. Despite much research on carotenoid biosynthesis in the model plant Arabidopsis thaliana, there is a lack of information on the carotenoid pathway in Brassica rapa. To better understand its carotenoid biosynthetic pathway, we performed a systematic analysis of carotenoid biosynthetic genes at the genome level in B. rapa.

Results

We identified 67 carotenoid biosynthetic genes in B. rapa, which were orthologs of the 47 carotenoid genes in A. thaliana. A high level of synteny was observed for carotenoid biosynthetic genes between A. thaliana and B. rapa. Out of 47 carotenoid biosynthetic genes in A. thaliana, 46 were successfully mapped to the 10 B. rapa chromosomes, and most of the genes retained more than one copy in B. rapa. The gene expansion was caused by the whole-genome triplication (WGT) event experienced by Brassica species. An expression analysis of the carotenoid biosynthetic genes suggested that their expression levels differed in root, stem, leaf, flower, callus, and silique tissues. Additionally, the paralogs of each carotenoid biosynthetic gene, which were generated from the WGT in B. rapa, showed significantly different expression levels among tissues, suggesting differentiated functions for these multi-copy genes in the carotenoid pathway.

Conclusions

This first systematic study of carotenoid biosynthetic genes in B. rapa provides insights into the carotenoid metabolic mechanisms of Brassica crops. In addition, a better understanding of carotenoid biosynthetic genes in B. rapa will contribute to the development of conventional and transgenic B. rapa cultivars with enriched carotenoid levels in the future.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1655-5) contains supplementary material, which is available to authorized users.     

Identification and comparative analysis of the Eriocheir sinensis microRNA transcriptome response to Spiroplasma eriocheiris infection using a deep sequencing approach

The Chinese mitten crab Eriocheir sinensis is one of the most important freshwater aquaculture crustacean species in China. MicroRNAs (miRNAs) are small non-coding RNAs that are important effectors in the intricate host-pathogen interaction network. To increase the repertoire of miRNAs characterized in crustaceans and to examine the relationship between host miRNA expression and pathogen infection, we used the Illumina/Solexa deep sequencing technology to sequence two small RNA libraries prepared from haemocytes of E. sinensis under normal conditions and during infection with Spiroplasma eriocheiris. The high-throughput sequencing resulted in approximately 30,975,151 and 30,826,277 raw reads corresponding to 12,077,088 and 16,271,545 high-quality mappable reads for the normal and infected haemocyte samples, respectively. Bioinformatic analyses identified 735 unique miRNAs, including 36 that are conserved in crustaceans, 134 that are novel to crabs but are present in other arthropods (PN-type), and 565 that are completely new (PC-type). Two hundred twenty-eight unique miRNAs displayed significant differential expression between the normal and infected haemocyte samples (p < 0.0001). Of these, 133 (58%) were significantly up-regulated and 95 (42%) were significantly down-regulated upon challenge with S. eriocheiris. Real-time quantitative PCR (RT-qPCR) experiments were preformed for 10 miRNAs of the two samples, and agreement was found between the sequencing and RT-qPCR data. To our knowledge, this is the first report of comprehensive identification of E. sinensis miRNAs and of expression analysis of E. sinensis miRNAs after exposure to S. eriocheiris. Many miRNAs were differentially regulated when exposed to the pathogen, and these findings support the hypothesis that certain miRNAs might be essential in host-pathogen interactions. Our results suggest that elucidation of the molecular mechanisms responsible for miRNA regulation of the host’s innate immune system should help with the development of new control strategies to prevent or treat S. eriocheiris infections in crustaceans.