PileLine: a toolbox to handle genome position information in next-generation sequencing studies

Background  

Genomic position (GP) files currently used in next-generation sequencing (NGS) studies are always difficult to manipulate due to their huge size and the lack of appropriate tools to properly manage them. The structure of these flat files is based on representing one line per position that has been covered by at least one aligned read, imposing significant restrictions from a computational performance perspective.     

Predation on northern shrimp (Pandalus borealis) by three gadoid species

Northern shrimp (Pandalus borealis) is targeted by commercial fisheries, but is also an important prey in the marine food web. In this study, stomach content data were used to study predation on shrimp by three gadoid species, cod (Gadus morhua), haddock (Melanogrammus aeglefinus) and whiting (Merlangius merlangus), in six inshore areas around Iceland. The results showed that shrimp was more important in the diet of cod compared with the other two predators. However, the overall predation pressure was similar for cod and haddock due to the high abundance of haddock. Therefore, even though shrimp is not the primary source of food for haddock, this species can have a substantial effect on shrimp stocks when haddock abundance is high. In addition, while cod and haddock did not select for any particular size of shrimp, whiting seemed to preferentially target juvenile shrimp. The results suggest that due to the overall effects of these three predators on shrimp stocks, gadoids need to be included in the management of shrimp stocks as predation is one of the major drivers in the development of this important prey stock.     

Predators of northern shrimp, Pandalus borealis (Pandalidae), throughout the North Atlantic

A compendium of northern shrimp (Pandalus borealis) predators was assembled, thereby consolidating decades of research that described trophic interactions among relevant marine species in the North Atlantic. The importance of shrimp as prey and the impacts of predation on shrimp populations were gleaned, where possible, from the literature. The review identified 26 species that prey on shrimp within North Atlantic ecosystems. The results clarified the role of shrimp as prey within these ecosystems, confirming that they provide an abundant source of food for marine fish, mammals and invertebrates throughout the North Atlantic. The evidence supported the likelihood of predation mortality as one of the key factors regulating shrimp population dynamics. However, lacking representative estimates of shrimp consumption by predators, the net effect on predation mortality within populations was unquantifiable.     

Amino acid sequence determination of a protein purified from the shell of the shrimp,Pandalus borealis

One of the urea-extractable proteins in the shell of the shrimp Pandalus borealis has been purified, and the complete amino acid sequence has been determined by the combined use of mass spectrometry and Edman degradation of the intact protein as well as of enzymatically derived peptides.Characteristic features of the sequence are discussed and compared to sequences from insect cuticular proteins and other structural proteins.     

Identification and characterization of vitellin in a hermophrodite shrimp,Pandalus kessleri

• 1.1. A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures.
• 2.2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively.
• 3.3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively.
• 4.4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.

     

Development of genomic simple sequence repeat markers in faba bean by next-generation sequencing

Faba bean (Vicia faba L.) is an important food legume crop with a huge genome. Development of genetic markers for faba bean is important to study diversity and for molecular breeding. In this study, we used Next Generation Sequencing (NGS) technology for the development of genomic simple sequence repeat (SSR) markers. A total of 14,027,500 sequence reads were obtained comprising 4,208 Mb. From these reads, 56,063 contigs were assembled (16,367 Mb) and 2138 SSRs were identified. Mono and dinucleotides were the most abundant, accounting for 57.5 % and 20.9 % of all SSR repeats, respectively. A total of 430 primer pairs were designed from contigs larger than 350 nucleotides and 50 primers pairs were tested for validation of SSR locus amplification. Nearly all (96 %) of the markers were found to produce clear amplicons and to be reproducible. Thirty-nine SSR markers were then applied to 46 faba bean accessions from worldwide origins, resulting in 161 alleles with 87.5 % polymorphism, and an average of 4.1 alleles per marker. Gene diversity (GD) of the markers ranged from 0 to 0.48 with an average of 0.27. Testing of the markers showed that they were useful in determining genetic relationships and population structure in faba bean accessions.     

Light and electron microscope study of a new species of Thelohania (Microsporida) in the shrimp Pandalus jordani

Thelohania butleri n. sp. was found in cells of skeletal muscles of the shrimp Pandalus jordani, from Queen Charlotte Sound, British Columbia, Canada. Sporulation stages were studied with the light and the electron microscope. Earliest stages were small and apparently uninucleate. Next were small diplokaryotic cells that possibly arose by fusion of the former. These enlarged and underwent sporogony. Sporogony was a series of three binary divisions, each producing unikaryotic cells. There was no sporogonial plasmodium. The spore was ovoid, 4.8 × 3.1 μm (stained), with a large crescentic nucleus and rounded posterior vacuole. The polar filament was isofilar, doubly coiled, with about 10 turns. This species closely resembles the type T. giardi Henneguy. It is concluded that sporogony by means of three binary divisions and lack of a sporogonial plasmodium may be essential characters of the genus Thelohania Henneguy, 1982.     

Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing

Opium poppy (Papaver somniferum L.) is an important pharmaceutical crop with very few genetic marker resources. To expand these resources, we sequenced genomic DNA using pyrosequencing technology and examined the DNA sequences for simple sequence repeats (SSRs). A total of 1,244,412 sequence reads were obtained covering 474 Mb. Approximately half of the reads (52 %) were assembled into 166,724 contigs representing 105 Mb of the opium poppy genome. A total of 23,283 non-redundant SSRs were identified in 18,944 contigs (11.3 % of total contigs). Trinucleotide and tetranucleotide repeats were the most abundant SSR repeats, accounting for 49.0 and 27.9 % of all SSRs, respectively. The AAG/TTC repeat was the most abundant trinucleotide repeat, representing 19.7 % of trinucleotide repeats. Other SSR repeat types were AT-rich. A total of 23,126 primer pairs (98.7 % of total SSRs) were designed to amplify SSRs. Fifty-three genomic SSR markers were tested in 37 opium poppy accessions and seven Papaver species for determination of polymorphism and transferability. Intraspecific polymorphism information content (PIC) values of the genomic SSR markers were intermediate, with an average 0.17, while the interspecific average PIC value was slightly higher, 0.19. All markers showed at least 88 % transferability among related species. This study increases sequence coverage of the opium poppy genome by sevenfold and the number of opium poppy-specific SSR markers by sixfold. This is the first report of the development of genomic SSR markers in opium poppy, and the genomic SSR markers developed in this study will be useful in diversity, identification, mapping and breeding studies in opium poppy.     

TILLING,high-resolution melting (HRM), and next-generation sequencing (NGS) techniques in plant mutation breeding

Induced mutations have been used effectively for plant improvement. Physical and chemical mutagens induce a high frequency of genome variation. Recently, developed screening methods have allowed the detection of single nucleotide polymorphisms (SNPs) and the identification of traits that are difficult to identify at the molecular level by conventional breeding. With the assistance of reverse genetic techniques, sequence variation information can be linked to traits to investigate gene function. Targeting induced local lesions in genomes (TILLING) is a high-throughput technique to identify single nucleotide mutations in a specific region of a gene of interest with a powerful detection method resulted from chemical-induced mutagenesis. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of genome size and ploidy level. However, TILLING requires laborious and time-consuming steps, and a lack of complete genome sequence information for many crop species has slowed the development of suitable TILLING targets. Another method, high-resolution melting (HRM), which has assisted TILLING in mutation detection, is faster, simpler and less expensive with non-enzymatic screening system. Currently, the sequencing of crop genomes has completely changed our vision and interpretation of genome organization and evolution. Impressive progress in next-generation sequencing (NGS) technologies has paved the way for the detection and exploitation of genetic variation in a given DNA or RNA molecule. This review discusses the applications of TILLING in combination with HRM and NGS technologies for screening of induced mutations and discovering SNPs in mutation breeding programs.     

Pathogen comparative genomics in the next-generation sequencing era: genome alignments, pangenomics and metagenomics

As soon as whole-genome sequencing entered the scene in the mid-1990s and demonstrated its use in revealing the entire genetic potential of any given microbial organism, this technique immediately revolutionized the way pathogen (and many other fields of) research was carried out. The ability to perform whole-genome comparisons further transformed the field and allowed scientists to obtain information linking phenotypic dissimilarities among closely related organisms and their underlying genetic mechanisms. Such comparisons have become commonplace in examining strain-to-strain variability, as well as comparing pathogens to less, or nonpathogenic near neighbors. In recent years, a bloom in novel sequencing technologies along with continuous increases in throughput has occurred, inundating the field with various types of massively parallel sequencing data and further transforming comparative genomics research. Here, we review the evolution of comparative genomics, its impact in understanding pathogen evolution and physiology and the opportunities and challenges presented by next-generation sequencing as applied to pathogen genome comparisons.     

Diversity and characterization of oxytetracycline-resistant bacteria associated with non-native species, white-leg shrimp (Litopenaeus vannamei), and native species, black tiger shrimp (Penaeus monodon), intensively cultured in Thailand

Aims: This study aimed at surveying prevalence of oxytetracycline (OTC)‐resistant bacteria in the white‐leg shrimp Litopenaeus vannamei, and the black tiger shrimp Penaeus monodon, intensively cultured in Thailand. We investigated the phylogenetic diversity of the bacterial isolates, as well as the minimum inhibitory concentration (MIC) of OTC, the occurrence of major OTC‐resistant genes and multiple‐antibiotic resistance in the isolates. Methods and Results: Shrimps were collected from culture ponds, and the homogenates of whole bodies were plated on tryptic soy agar supplemented with or without OTC. Percentages of OTC‐resistant bacteria were 0·3–52·1% in white‐leg samples and 0·008–22·3% in black tiger samples. Analyses of 16S rDNA sequences indicated that most OTC‐resistant isolates were closely related to Aeromonas spp. and Lactococcus garvieae. MICs of OTC were 4–128 μg ml^?1 in the OTC‐resistant aeromonads and 128–256 μg ml^?1 in OTC‐resistant L. garvieae. OTC resistance was found to be conferred by the genes tet(A), tet(C), tet(D), tet(E), tet(M) and tet(S), detected either singly or in pairs. No resistance to ceftazidime, imipenem or chloramphenicol was observed in any isolate. Conclusions: Both species of shrimp are associated with OTC‐resistant bacteria, occasionally at high densities exceeding 10⁶ cfu g^?1. The associated bacteria, predominantly Lactococcus and Aeromonas genera, are potential pathogens and are reservoirs of a variety of OTC‐resistant genes. Significance and Impact of the Study: Cultured shrimps can be vehicle to carry OTC‐resistant bacteria to domestic and foreign consumers via the food chain. Very low populations of OTC‐resistant bacteria observed in the several ponds suggest that levels of the resistant bacteria are artificially high and should be reduced in farmed shrimps.     

The use of genotyping by sequencing in blackcurrant (Ribes nigrum): developing high-resolution linkage maps in species without reference genome sequences

A genotyping by sequencing (GbS) approach is reported in blackcurrant (Ribes nigrum L.) using a de novo read assembly method developed because of the current absence of a reference genome sequence for this species. A new approach to single nucleotide polymorphism (SNP) genotype calling is described, where individual genotypes for a large number of SNPs were characterised from the GbS counts using a novel method based on a functional regression of major and minor allele read counts. The high-quality GbS SNPs were combined with SNPs and simple sequence repeats generated from other technologies to develop a linkage map with increased marker density and improved genome coverage, containing up to 204 SNPs on each linkage group. SNPs of lower quality were then located on the map using quantitative trait locus (QTL) interval mapping of the proportion of the major allele. Two QTL each for 100-berry weight and Brix scores, measured over three years, were identified using the map. The use of this approach to identify and map a significant number of novel SNPs in a woody species with hitherto limited genomic resources may have generic application to other under-resourced and minor species in the development of cost-effective and efficient high-density genetic maps.     

Interactions between northern shrimp, Pandalus borealis (Pandalidae), and its key predators within the eastern Newfoundland and Labrador marine ecosystem

An examination of population trends for northern shrimp (Pandalus borealis) and its key predators off eastern Newfoundland and Labrador provided the basis for investigating predator-prey relationships within an ecosystem that experienced major changes in species composition during the 1980s and 1990s. Populations of several demersal fish species, known to feed on northern shrimp, declined to historically low levels by the early 1990s and remained depressed thereafter. Some declines were precipitous from the late 1980s to early 1990s, coincident with an increase in shrimp. Populations of other important predator species increased throughout the 1990s along with shrimp. Lacking representative estimates of shrimp consumption, the net effect on predation mortality was unquantifiable and it was not possible to demonstrate with certainty that the major increase in shrimp biomass that occurred throughout the 1990s resulted from a concomitant reduction in predation mortality. Factors (and their interactions) relevant to the dynamics of shrimp populations include the effects of ocean climate, predation, competition, as well as commercial harvesting.