Characterization of miRNAs responsive to exogenous ethylene in grapevine berries at whole genome level

MicroRNAs (miRNAs) are critical regulators of various biological and metabolic processes of plants. Numerous miRNAs and their functions have been identified and analyzed in many plants. However, till now, the involvement of miRNAs in the response of grapevine berries to ethylene has not been reported yet. Here, Solexa technology was employed to deeply sequence small RNA libraries constructed from grapevine berries treated with and without ethylene. A total of 124 known and 78 novel miRNAs were identified. Among these miRNAs, 162 miRNAs were clearly responsive to ethylene, with 55 downregulated, 59 upregulated, and 14 unchanged miRNAs detected only in the control. The other 35 miRNAs responsive to ethylene were induced by ethylene and detected only in the ethylene-treated grapevine materials. Expression analysis of 27 conserved and 26 novel miRNAs revealed that 13 conserved and 18 novel ones were regulated by ethylene during the whole development of grapevine berries. High-throughput sequencing and qRT-PCR assays revealed consistent results on the expression results of ethylene-responsive miRNAs. Moreover, 90 target genes for 34 novel miRNAs were predicted, most of which were involved in responses to various stresses, especially like exogenous ethylene treatment. The identified miRNAs may be mainly involved in grapevine berry development and response to various environmental conditions.     

Characterizing homologues of crop domestication genes in poorly described wild relatives by high‐throughput sequencing of whole genomes

Wild crop relatives represent a source of novel alleles for crop genetic improvement. Screening biodiversity for useful or diverse gene homologues has often been based upon the amplification of targeted genes using available sequence information to design primers that amplify the target gene region across species. The crucial requirement of this approach is the presence of sequences with sufficient conservation across species to allow for the design of universal primers. This approach is often not successful with diverse organisms or highly variable genes. Massively parallel sequencing (MPS) can quickly produce large amounts of sequence data and provides a viable option for characterizing homologues of known genes in poorly described genomes. MPS of genomic DNA was used to obtain species‐specific sequence information for 18 rice genes related to domestication characteristics in a wild relative of rice, Microlaena stipoides. Species‐specific primers were available for 16 genes compared with 12 genes using the universal primer method. The use of species‐specific primers had the potential to cover 92% of the sequence of these genes, while traditional universal primers could only be designed to cover 80%. A total of 24 species‐specific primer pairs were used to amplify gene homologues, and 11 primer pairs were successful in capturing six gene homologues. The 23 million, 36‐base pair (bp) paired end reads, equated to an average of 2X genome coverage, facilitated the successful amplification and sequencing of six target gene homologues, illustrating an important approach to the discovery of useful genes in wild crop relatives.     

Introgression browser: high‐throughput whole‐genome SNP visualization

Breeding by introgressive hybridization is a pivotal strategy to broaden the genetic basis of crops. Usually, the desired traits are monitored in consecutive crossing generations by marker‐assisted selection, but their analyses fail in chromosome regions where crossover recombinants are rare or not viable. Here, we present the Introgression Browser (iBrowser ), a bioinformatics tool aimed at visualizing introgressions at nucleotide or SNP (Single Nucleotide Polymorphisms) accuracy. The software selects homozygous SNPs from Variant Call Format (VCF) information and filters out heterozygous SNPs, multi‐nucleotide polymorphisms (MNPs) and insertion–deletions (InDels). For data analysis iBrowser makes use of sliding windows, but if needed it can generate any desired fragmentation pattern through General Feature Format (GFF) information. In an example of tomato (Solanum lycopersicum) accessions we visualize SNP patterns and elucidate both position and boundaries of the introgressions. We also show that our tool is capable of identifying alien DNA in a panel of the closely related S. pimpinellifolium by examining phylogenetic relationships of the introgressed segments in tomato. In a third example, we demonstrate the power of the iBrowser in a panel of 597 Arabidopsis accessions, detecting the boundaries of a SNP‐free region around a polymorphic 1.17 Mbp inverted segment on the short arm of chromosome 4. The architecture and functionality of iBrowser makes the software appropriate for a broad set of analyses including SNP mining, genome structure analysis, and pedigree analysis. Its functionality, together with the capability to process large data sets and efficient visualization of sequence variation, makes iBrowser a valuable breeding tool.     

Digital fragment analysis of short tandem repeats by high‐throughput amplicon sequencing

High‐throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high‐throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high‐throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost‐effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence‐aware microsatellite data.     

Promises and pitfalls of using high‐throughput sequencing for diet analysis

The application of high‐throughput sequencing‐based approaches to DNA extracted from environmental samples such as gut contents and faeces has become a popular tool for studying dietary habits of animals. Due to the high resolution and prey detection capacity they provide, both metabarcoding and shotgun sequencing are increasingly used to address ecological questions grounded in dietary relationships. Despite their great promise in this context, recent research has unveiled how a wealth of biological (related to the study system) and technical (related to the methodology) factors can distort the signal of taxonomic composition and diversity. Here, we review these studies in the light of high‐throughput sequencing‐based assessment of trophic interactions. We address how the study design can account for distortion factors, and how acknowledging limitations and biases inherent to sequencing‐based diet analyses are essential for obtaining reliable results, thus drawing appropriate conclusions. Furthermore, we suggest strategies to minimize the effect of distortion factors, measures to increase reproducibility, replicability and comparability of studies, and options to scale up DNA sequencing‐based diet analyses. In doing so, we aim to aid end‐users in designing reliable diet studies by informing them about the complexity and limitations of DNA sequencing‐based diet analyses, and encourage researchers to create and improve tools that will eventually drive this field to its maturity.     

Prevention,diagnosis and treatment of high‐throughput sequencing data pathologies

High‐throughput sequencing (HTS) technologies generate millions of sequence reads from DNA/RNA molecules rapidly and cost‐effectively, enabling single investigator laboratories to address a variety of ‘omics’ questions in nonmodel organisms, fundamentally changing the way genomic approaches are used to advance biological research. One major challenge posed by HTS is the complexity and difficulty of data quality control (QC). While QC issues associated with sample isolation, library preparation and sequencing are well known and protocols for their handling are widely available, the QC of the actual sequence reads generated by HTS is often overlooked. HTS‐generated sequence reads can contain various errors, biases and artefacts whose identification and amelioration can greatly impact subsequent data analysis. However, a systematic survey on QC procedures for HTS data is still lacking. In this review, we begin by presenting standard ‘health check‐up’ QC procedures recommended for HTS data sets and establishing what ‘healthy’ HTS data look like. We next proceed by classifying errors, biases and artefacts present in HTS data into three major types of ‘pathologies’, discussing their causes and symptoms and illustrating with examples their diagnosis and impact on downstream analyses. We conclude this review by offering examples of successful ‘treatment’ protocols and recommendations on standard practices and treatment options. Notwithstanding the speed with which HTS technologies – and consequently their pathologies – change, we argue that careful QC of HTS data is an important – yet often neglected – aspect of their application in molecular ecology, and lay the groundwork for developing a HTS data QC ‘best practices’ guide.     

Diversity and dynamics of microbial communities in brown planthopper at different developmental stages revealed by high‐throughput amplicon sequencing

The microbiome associated with brown planthopper (BPH) plays an important role in mediating host health and fitness. Characterization of the microbial community and its structure is prerequisite for understanding the intricate symbiotic relationships between microbes and host insect. Here, we investigated the bacterial and fungal communities of BPH at different developmental stages using high‐throughput amplicon sequencing. Our results revealed that both the bacterial and fungal communities were diverse and dynamic during BPH development. The bacterial communities were generally richer than fungi in each developmental stage. At 97% similarly, 19 phyla and 278 genera of bacteria were annotated, while five fungal phyla comprising 80 genera were assigned. The highest species richness for the bacterial communities was detected in the nymphal stage. The taxonomic diversity of the fungal communities in female adults was generally at a relatively higher level when compared to other developmental stages. The most dominant phylum of bacteria and fungi at each developmental stage all belonged to Proteobacteria and Ascomycota, respectively. A significantly lower abundance of bacterial genus Acinetobacter was recorded in the egg stage when compared to other developmental stages, while the dominant fungal genus Wallemia was more abundant in the nymph and adult stages than in the egg stage. Additionally, the microbial composition differed between male and female adults, suggesting that the microbial communities in BPH were gender‐dependent. Overall, our study enriches our knowledge on the microbial communities associated with BPH and will provide clues to develop potential biocontrol techniques against this rice pest.     

jackalope: A swift,versatile phylogenomic and high‐throughput sequencing simulator

High‐throughput sequencing (HTS) is central to the study of population genomics and has an increasingly important role in constructing phylogenies. Choices in research design for sequencing projects can include a wide range of factors, such as sequencing platform, depth of coverage and bioinformatic tools. Simulating HTS data better informs these decisions, as users can validate software by comparing output to the known simulation parameters. However, current standalone HTS simulators cannot generate variant haplotypes under even somewhat complex evolutionary scenarios, such as recombination or demographic change. This greatly reduces their usefulness for fields such as population genomics and phylogenomics. Here I present the R package jackalope that simply and efficiently simulates (i) sets of variant haplotypes from a reference genome and (ii) reads from both Illumina and Pacific Biosciences platforms. Haplotypes can be simulated using phylogenies, gene trees, coalescent‐simulation output, population‐genomic summary statistics, and Variant Call Format (VCF) files. jackalope can simulate single, paired‐end or mate‐pair Illumina reads, as well as reads from Pacific Biosciences. These simulations include sequencing errors, mapping qualities, multiplexing and optical/PCR duplicates. It can read reference genomes from fasta files and can simulate new ones, and all outputs can be written to standard file formats. jackalope is available for Mac, Windows and Linux systems.     

Applying high‐throughput sequencing to identify and evaluate foetal chromosomal deletion and duplication

The present study aimed to estimate the clinical performance of non‐invasive prenatal testing (NIPT) based on high‐throughput sequencing method for the detection of foetal chromosomal deletions and duplications. A total of 6348 pregnant women receiving NIPT using high‐throughput sequencing method were included in our study. They all conceived naturally, without twins, triplets or multiple births. Individuals showing abnormalities in NIPT received invasive ultrasound‐guided amniocentesis for chromosomal karyotype and microarray analysis at 18‐24 weeks of pregnancy. Detection results of foetal chromosomal deletions and duplications were compared between high‐throughput sequencing method and chromosomal karyotype and microarray analysis. Thirty‐eight individuals were identified to show 51 chromosomal deletions/duplications via high‐throughput sequencing method. In subsequent chromosomal karyotype and microarray analysis, 34 subchromosomal deletions/duplications were identified in 26 pregnant women. The observed deletions and duplications ranged from 1.05 to 17.98 Mb. Detection accuracy for these deletions and duplications was 66.7%. Twenty‐one deletions and duplications were found to be correlated with the known abnormalities. NIPT based on high‐throughput sequencing technique is able to identify foetal chromosomal deletions and duplications, but its sensitivity and specificity were not explored. Further progress should be made to reduce false‐positive results.     

Modular tagging of amplicons using a single PCR for high‐throughput sequencing

High‐throughput sequencing (HTS) of PCR amplicons is becoming the method of choice to sequence one or several targeted loci for phylogenetic and DNA barcoding studies. Although the development of HTS has allowed rapid generation of massive amounts of DNA sequence data, preparing amplicons for HTS remains a rate‐limiting step. For example, HTS platforms require platform‐specific adapter sequences to be present at the 5′ and 3′ end of the DNA fragment to be sequenced. In addition, short multiplex identifier (MID) tags are typically added to allow multiple samples to be pooled in a single HTS run. Existing methods to incorporate HTS adapters and MID tags into PCR amplicons are either inefficient, requiring multiple enzymatic reactions and clean‐up steps, or costly when applied to multiple samples or loci (fusion primers). We describe a method to amplify a target locus and add HTS adapters and MID tags via a linker sequence using a single PCR. We demonstrate our approach by generating reference sequence data for two mitochondrial loci (COI and 16S) for a diverse suite of insect taxa. Our approach provides a flexible, cost‐effective and efficient method to prepare amplicons for HTS.     

Seasonal diversity and dynamics of haptophytes in the Skagerrak,Norway, explored by high‐throughput sequencing

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high‐throughput sequencing. From outer Oslofjorden, S Norway, nano‐ and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta‐specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA‐sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September–October (autumn) and lowest in April–May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP‐3–5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in‐depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters.     

High‐throughput sequencing of Hop stunt viroid‐derived small RNAs from cucumber leaves and phloem

Small RNA (sRNA)‐guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self‐replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of viroid‐derived sRNAs (vd‐sRNAs) is usually associated with viroid infection. However, the pathways involved in the biogenesis of vd‐sRNAs are largely unknown. Here, we analyse, by high‐throughput pyrosequencing, the profiling of the Hop stunt viroid (HSVd) vd‐sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd‐sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd‐sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd‐sRNAs. Our sequence data suggest that viroid‐derived double‐stranded RNA functions as one of the main precursors of vd‐sRNAs. Remarkably, phloem vd‐sRNAs accumulated preferentially as 22‐nucleotide species with a consensus sequence over‐represented. This bias in size and sequence in the HSVd vd‐sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd‐sRNAs to the phloem tissue of infected cucumber plants.     

High‐throughput SNP discovery in the rabbit (Oryctolagus cuniculus) genome by next‐generation semiconductor‐based sequencing

The European rabbit (Oryctolagus cuniculus) is a domesticated species with one of the broadest ranges of economic and scientific applications and fields of investigation. Rabbit genome information and assembly are available (oryCun2.0), but so far few studies have investigated its variability, and massive discovery of polymorphisms has not been published yet for this species. Here, we sequenced two reduced representation libraries (RRLs) to identify single nucleotide polymorphisms (SNPs) in the rabbit genome. Genomic DNA of 10 rabbits belonging to different breeds was pooled and digested with two restriction enzymes (HaeIII and RsaI) to create two RRLs which were sequenced using the Ion Torrent Personal Genome Machine. The two RRLs produced 2 917 879 and 4 046 871 reads, for a total of 280.51 Mb (248.49 Mb with quality >20) and 417.28 Mb (360.89 Mb with quality >20) respectively of sequenced DNA. About 90% and 91% respectively of the obtained reads were mapped on the rabbit genome, covering a total of 15.82% of the oryCun2.0 genome version. The mapping and ad hoc filtering procedures allowed to reliably call 62 491 SNPs. SNPs in a few genomic regions were validated by Sanger sequencing. The Variant Effect Predictor Web tool was used to map SNPs on the current version of the rabbit genome. The obtained results will be useful for many applied and basic research programs for this species and will contribute to the development of cost‐effective solutions for high‐throughput SNP genotyping in the rabbit.     

Comparing the performance of three ancient DNA extraction methods for high‐throughput sequencing

The DNA molecules that can be extracted from archaeological and palaeontological remains are often degraded and massively contaminated with environmental microbial material. This reduces the efficacy of shotgun approaches for sequencing ancient genomes, despite the decreasing sequencing costs of high‐throughput sequencing (HTS). Improving the recovery of endogenous molecules from the DNA extraction and purification steps could, thus, help advance the characterization of ancient genomes. Here, we apply the three most commonly used DNA extraction methods to five ancient bone samples spanning a ~30 thousand year temporal range and originating from a diversity of environments, from South America to Alaska. We show that methods based on the purification of DNA fragments using silica columns are more advantageous than in solution methods and increase not only the total amount of DNA molecules retrieved but also the relative importance of endogenous DNA fragments and their molecular diversity. Therefore, these methods provide a cost‐effective solution for downstream applications, including DNA sequencing on HTS platforms.