Sufficient Sequence Research Articles

Impact of sequencing depth in ChIP-seq experiments

In a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiment, an important consideration in experimental design is the minimum number of sequenced reads required to obtain statistically significant results. We present an extensive evaluation of the impact of sequencing depth on identification of enriched regions for key histone modifications (H3K4me3, H3K36me3, H3K27me3 and H3K9me2/me3) using deep-sequenced datasets in human and fly. We propose to define sufficient sequencing depth as the number of reads at which detected enrichment regions increase <1% for an additional million reads. Although the required depth depends on the nature of the mark and the state of the cell in each experiment, we observe that sufficient depth is often reached at <20 million reads for fly. For human, there are no clear saturation points for the examined datasets, but our analysis suggests 40–50 million reads as a practical minimum for most marks. We also devise a mathematical model to estimate the sufficient depth and total genomic coverage of a mark. Lastly, we find that the five algorithms tested do not agree well for broad enrichment profiles, especially at lower depths. Our findings suggest that sufficient sequencing depth and an appropriate peak-calling algorithm are essential for ensuring robustness of conclusions derived from ChIP-seq data.

Inhalable Microorganisms in Beijing’s PM2.5 and PM10 Pollutants during a Severe Smog Event

Particulate matter (PM) air pollution poses a formidable public health threat to the city of Beijing. Among the various hazards of PM pollutants, microorganisms in PM2.5 and PM10 are thought to be responsible for various allergies and for the spread of respiratory diseases. While the physical and chemical properties of PM pollutants have been extensively studied, much less is known about the inhalable microorganisms. Most existing data on airborne microbial communities using 16S or 18S rRNA gene sequencing to categorize bacteria or fungi into the family or genus levels do not provide information on their allergenic and pathogenic potentials. Here we employed metagenomic methods to analyze the microbial composition of Beijing’s PM pollutants during a severe January smog event. We show that with sufficient sequencing depth, airborne microbes including bacteria, archaea, fungi, and dsDNA viruses can be identified at the species level. Our results suggested that the majority of the inhalable microorganisms were soil-associated and nonpathogenic to human. Nevertheless, the sequences of several respiratory microbial allergens and pathogens were identified and their relative abundance appeared to have increased with increased concentrations of PM pollution. Our findings may serve as an important reference for environmental scientists, health workers, and city planners.

Characterization of the house sparrow (Passer domesticus) transcriptome: a resource for molecular ecology and immunogenetics

The house sparrow (Passer domesticus) is an important model species in ecology and evolution. However, until recently, genomic resources for molecular ecological projects have been lacking in this species. Here, we present transcriptome sequencing data (RNA-Seq) from three different house sparrow tissues (spleen, blood and bursa). These tissues were specifically chosen to obtain a diverse representation of expressed genes and to maximize the yield of immune-related gene functions. After de novo assembly, 15,250 contigs were identified, representing sequence data from a total of 8756 known avian genes (as inferred from the closely related zebra finch). The transcriptome assembly contain sequence data from nine manually annotated MHC genes, including an almost complete MHC class I coding sequence. There were 407, 303 and 68 genes overexpressed in spleen, blood and bursa, respectively. Gene ontology terms related to ribosomal function were associated with overexpression in spleen and oxygen transport functions with overexpression in blood. In addition to the transcript sequences, we provide 327 gene-linked microsatellites (SSRs) with sufficient flanking sequences for primer design, and 3177 single-nucleotide polymorphisms (SNPs) within genes, that can be used in follow-up molecular ecology studies of this ecological well-studied species.

Cluster based prediction of PDZ-peptide interactions

BackgroundPDZ domains are one of the most promiscuous protein recognition modules that bind with short linear peptides and play an important role in cellular signaling. Recently, few high-throughput techniques (e.g. protein microarray screen, phage display) have been applied to determine in-vitro binding specificity of PDZ domains. Currently, many computational methods are available to predict PDZ-peptide interactions but they often provide domain specific models and/or have a limited domain coverage.ResultsHere, we composed the largest set of PDZ domains derived from human, mouse, fly and worm proteomes and defined binding models for PDZ domain families to improve the domain coverage and prediction specificity. For that purpose, we first identified a novel set of 138 PDZ families, comprising of 548 PDZ domains from aforementioned organisms, based on efficient clustering according to their sequence identity. For 43 PDZ families, covering 226 PDZ domains with available interaction data, we built specialized models using a support vector machine approach. The advantage of family-wise models is that they can also be used to determine the binding specificity of a newly characterized PDZ domain with sufficient sequence identity to the known families. Since most current experimental approaches provide only positive data, we have to cope with the class imbalance problem. Thus, to enrich the negative class, we introduced a powerful semi-supervised technique to generate high confidence non-interaction data. We report competitive predictive performance with respect to state-of-the-art approaches.ConclusionsOur approach has several contributions. First, we show that domain coverage can be increased by applying accurate clustering technique. Second, we developed an approach based on a semi-supervised strategy to get high confidence negative data. Third, we allowed high order correlations between the amino acid positions in the binding peptides. Fourth, our method is general enough and will easily be applicable to other peptide recognition modules such as SH2 domains and finally, we performed a genome-wide prediction for 101 human and 102 mouse PDZ domains and uncovered novel interactions with biological relevance. We make all the predictive models and genome-wide predictions freely available to the scientific community.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-S1-S5) contains supplementary material, which is available to authorized users.

Restriction enzyme-mediated DNA family shuffling.

DNA shuffling is an established recombinatorial method that was originally developed to increase the speed of directed evolution experiments beyond what could be accomplished using error-prone PCR alone. To achieve this, mutated copies of a protein-coding sequence are fragmented with DNase I and the fragments are then reassembled in a PCR without primers. The fragments anneal where there is sufficient sequence identity, resulting in full-length variants of the original gene that have inherited mutations from multiple templates. Subsequent studies demonstrated that directed evolution could be further accelerated by shuffling similar native protein-coding sequences from the same gene family, rather than mutated variants of a single gene. Generally at least 65-75 % global identity between parental sequences is required in DNA family shuffling, with recombination mostly occurring at sites with at least five consecutive nucleotides of local identity. Since DNA shuffling was originally developed, many variations on the method have been published. In particular, the use of restriction enzymes in the fragmentation step allows for greater customization of fragment lengths than DNase I digestion and avoids the risk that parental sequences may be over-digested into unusable very small fragments. Restriction enzyme-mediated fragmentation also reduces the occurrence of undigested parental sequences that would otherwise reduce the number of unique variants in the resulting library. In the current chapter, we provide a brief overview of the alternative methods currently available for DNA shuffling as well as a protocol presented here that improves on several previous implementations of restriction enzyme-mediated DNA family shuffling, in particular with regard to purification of DNA fragments for reassembly.

Species delimitation of the genus Champia (Rhodymeniales, Rhodophyta) from Korea using DNA barcoding

DNA barcoding is becoming a widely applied tool to accurately discriminate red algae. We tested the effectiveness of DNA barcoding for identification and discovery of Champia species in Korea and clarified the phylogenetic relationships using the plastid rbcL gene. As results, we described four species of Champia such as C. inkyua sp. nov., C. recta Noda, C. bifida Okamura, and C. expansa Yendo. A new species, C. inkyua, is characterized by entangled thallus, terete and irregular branches, hooked apices, and longitudinal filaments running throughout the frond periphery only. Longitudinal filaments were composed of a complete cell with two half cells between diaphragms in the cavity. C. recta and C. bifida were reinstated with previously used names of C. parvula and C. compressa, respectively. C. recta is the first recorded species from Korea and is characterized by an erect thallus, terete and irregular branches, and straight apices. C. bifida is characterized by compressed thallus, pinnate or alternate branches, and bifid apices. C. expansa is characterized by flabellate thallus and dichotomous branches. Molecular analyses of COI and rbcL genes revealed sufficient sequence divergence to warrant species recognition in the genus Champia.

Rapid identification of Zygosaccharomyces with genus-specific primers

There has been a recent and rapid increase in the number of species of the genus Zygosaccharomyces which now comprises Z. bailii, Z. bisporus, Z. gambellarensis, Z. kombuchaensis, Z. lentus, Z. machadoi, Z. mellis, Z. parabaillii, Z. pseudobailii, Z. pseudorouxii, Z. rouxii, Z. sapae, and Z. siamensis. Z. pseudorouxii is an unofficial name given to isolates closely related to the newly-described species Z. sapae. The Zygosaccharomyces genus contains species that are important as food and beverage spoilage organisms and others are associated with fermentations and sweet foodstuffs, such as honey. Their economic significance means that the ability to identify them rapidly is of significant importance. Although Z. rouxii and Z. bailii have been genome-sequenced the extent of sequence data for the others, especially the newly-discovered species, is sometimes extremely limited which makes identification slow. However, parts of the ITS1/5.8S/ITS2 rDNA region contain sequences of sufficient similarity within the genus and of sufficient difference with outgroups, to be potential regions for the design of genus-wide specific primers. We report here the development of genus-specific primers that can detect all the major Zygosaccharomyces species including all those associated with foods; the rare and localised species Z. machadoi and Z. gambellarensis are not detected. The size of the single amplicon produced varies between species and in some cases is sufficiently different to assign provisional species identification. Sequence data from rDNA regions are available for virtually all described yeast species in all genera, thus, prior to having sufficient sequence data from structural genes, rDNA regions may provide more generally suitable candidates for both genus-specific and species-specific primer design.

Genome-Wide Microsatellite Identification in the Fungus Anisogramma anomala Using Illumina Sequencing and Genome Assembly

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N50 of 10,384 bp and scaffold N50 of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery.

Association of single nucleotide polymorphisms(SNPs) in Pygo2 coding gene with idiopathic oligospermia and azoospermia

Male infertility is often associated with a decreased sperm count. Pygo2 gene is expressed in the elongating spermatid when chromatin remodeling occurs, thus it is possible that impairment of Pygo2 function could lead to spermatogenic arrest, reduction of sperm count and subsequent infertility. The aim of this study was to detect mutations in Pygo2 that lead to idiopathic oligospermia and azoospermia in human. DNA was isolated from venous blood from 77 fertile and 195 idiopathic oligospermic or azoospermic men. PCR-sequencing analysis was performed for the 3 coding regions of Pygo2. Non-synonymous single nucleotide polymorphisms (SNPs) were detected and analyzed using SIFT, Polyphen-2 and Mutation Taster software to determine possible changes in protein structure that could affect phenotype. Of the 195 patients analyzed, sufficient gene sequencing was accomplished for 178 men (30 mild or moderate oligospermic, 57 severe oligospermic and 91 azoospermic men). Three previously reported non-synonymous SNPs were identified in azoospermic and severe oligospermic patients and not in mild and moderate oligozoopermic or normozoospermic men. SNP rs61758740 (M141I) causes the replacement of a hydrophobic amino acid with another hydrophobic amino acid, rs61758741 (K261E) causes the replacement of a basic amino acid with an acidic amino acid and rs141722381 (N261I) causes the replacement of a hydrophilic amino acid with another hydrophobic amino acid. The data predicted by three different software programs showed that SNP rs141722381 results in the damage of tertiary protein structure and thus could be involved in relevant diseases. The study demonstrates that SNPs in the coding region of Pygo2 gene may be one of the causative factors in idiopathic oligospermia and azoospermia, resulting in male infertility.

Targeted next-generation sequencing reveals further genetic heterogeneity in axonal Charcot–Marie–Tooth neuropathy and a mutation in HSPB1

Charcot-Marie-Tooth disease (CMT) is a group of hereditary peripheral neuropathies. The dominantly inherited axonal CMT2 displays striking genetic heterogeneity, with 17 presently known disease genes. The large number of candidate genes, combined with lack of genotype-phenotype correlations, has made genetic diagnosis in CMT2 time-consuming and costly. In Finland, 25% of dominant CMT2 is explained by either a GDAP1 founder mutation or private MFN2 mutations but the rest of the families have remained without molecular diagnosis. Whole-exome and genome sequencing are powerful techniques to find disease mutations for CMT patients but they require large amounts of sequencing to confidently exclude heterozygous variants in all candidate genes, and they generate a vast amount of irrelevant data for diagnostic needs. Here we tested a targeted next-generation sequencing approach to screen the CMT2 genes. In total, 15 unrelated patients from dominant CMT2 families from Finland, in whom MFN2 and GDAP1 mutations had been excluded, participated in the study. The targeted approach produced sufficient sequence coverage for 95% of the 309 targeted exons, the rest we excluded by Sanger sequencing. Unexpectedly, the screen revealed a disease mutation only in one family, in the HSPB1 gene. Thus, new disease genes underlie CMT2 in the remaining families, indicating further genetic heterogeneity. We conclude that targeted next-generation sequencing is an efficient tool for genetic screening in CMT2 that also aids in the selection of patients for genome-wide approaches.

DNA barcoding the commercial Chinese caterpillar fungus

Chinese caterpillar fungus (Ophiocordyceps sinensis) has been widely used as tonic in Asian medicine. Considering its curative effect and high cost, various counterfeit versions of O. sinensis have been introduced and are commercially available. These counterfeits have morphological characteristics that are difficult to distinguish based on morphology alone, thereby causing confusion and threatening its safe use. In this study, internal transcribed spacer (ITS) sequences as a DNA barcode were analyzed and assessed for rapid and accurate identification of 131 O. sinensis samples and 12 common counterfeits and closely related species. Results showed that sufficient ITS sequence differences, also known as 'barcode gaps', existed to distinguish between O. sinensis and counterfeit species. ITS sequence correctly identified 100% of the samples at the species and genus level using the Basic Local Alignment Search Tool 1 and the nearest distance method. Furthermore, O. sinensis, counterfeits, and closely related species can be successfully identified using tree-based methods including maximum parsimony, neighbor-joining, and maximum likelihood analysis. These results indicated that DNA barcoding could be used as a fast and accurate identification method to distinguish O. sinensis from counterfeits and closely related species to ensure its safe use.

Development of 25 Novel Microsatellite Loci and Genetic Variation Analysis in Breeding Populations of the Pearl Oyster,Pinctada fucata

AbstractIn this study, we screened 103,050 gene sequences ofPinctada fucataand identified 1450 simple sequence repeats (SSRs) distributing in 1355 sequences, in which 97 have sufficient flanking sequences for primer design, and 25 novel microsatellite loci were stable and polymorphic withNawhich varied from 2 to 6. Six microsatellite loci were found in the open reading frame of their corresponding sequences. With the 25SSRloci, five breeding populations and a wild population, a total of 240 individuals from wild population (W), three consecutive selected populations (G1,G2,andG3), and two backcross populations (Do, Dr) (40 individuals per population) were assayed. In these populations, we detected 108 alleles of 111 bp to 362 bp. Each population had meanHoof 0.5718–0.7366 and meanHeof 0.5830–0.6954. Except theFst = 0.0697 (W vs.G3), pairwiseFstvalues ranged from 0.0131 to 0.0473, the genetic identity coefficients varied from 0.8688 to 0.9663, and genetic distance ranged from 0.0343 to 0.1307, all of these suggested that the breeding populations had genetic differentiation but at a low level generally. Besides, all the genetic index values, backcross populations (Do, Dr) were higher than correspondingG3values, indicating that backcross could make the offspring incline to the recurrent parents.

A systematic evaluation of hybridization-based mouse exome capture system

BackgroundExome sequencing is increasingly used to search for phenotypically-relevant sequence variants in the mouse genome. All of the current hybridization-based mouse exome capture systems are designed based on the genome reference sequences of the C57BL/6 J strain. Given that the substantial sequence divergence exists between C57BL/6 J and other distantly-related strains, the impact of sequence divergence on the efficiency of such capture systems needs to be systematically evaluated before they can be widely applied to the study of those strains.ResultsUsing the Agilent SureSelect mouse exome capture system, we performed exome sequencing on F1 generation hybrid mice that were derived by crossing two divergent strains, C57BL/6 J and SPRET/EiJ. Our results showed that the C57BL/6 J-based probes captured the sequences derived from C57BL/6 J alleles more efficiently and that the bias was higher for the target regions with greater sequence divergence. At low sequencing depths, the bias also affected the efficiency of variant detection. However, the effects became negligible when sufficient sequencing depth was achieved.ConclusionSufficient sequence depth needs to be planned to match the sequence divergence between C57BL/6 J and the strain to be studied, when the C57BL/6 J–based Agilent SureSelect exome capture system is to be used.

Does germ-line deletion of the PIP gene constitute a widespread risk for cancer?

Does germ-line deletion of the PIP gene constitute a widespread risk for cancer?

Characterization of 582 natural and synthetic terminators and quantification of their design constraints

Large genetic engineering projects require more cistrons and consequently more strong and reliable transcriptional terminators. We have measured the strengths of a library of terminators, including 227 that are annotated in Escherichia coli--90 of which we also tested in the reverse orientation--and 265 synthetic terminators. Within this library we found 39 strong terminators, yielding >50-fold reduction in downstream expression, that have sufficient sequence diversity to reduce homologous recombination when used together in a design. We used these data to determine how the terminator sequence contributes to its strength. The dominant parameters were incorporated into a biophysical model that considers the role of the hairpin in the displacement of the U-tract from the DNA. The availability of many terminators of varying strength, as well as an understanding of the sequence dependence of their properties, will extend their usability in the forward design of synthetic cistrons.

Abstract 3509: Complementary analysis of LKB1/STK11 mutation and protein expression status using next-generation sequencing, Sanger sequencing and immunohistochemistry.

Abstract Introduction: LKB1 (liver kinase B1)/STK11 (serine-threonine kinase 11) is a tumor suppressor that encodes a serine/threonine kinase which negatively regulates the mTOR (mammalian target of rapamycin) signaling pathway. Somatic mutations in LKB1 occur most notably in lung adenocarcinoma, cervical cancers and melanoma. Inactivation of LKB1 is caused by point mutations, homozygous deletions or promoter methylation. LKB1 mutation status is a predictive marker for responsiveness to both MEK and PI3K inhibitors. We have developed next-generation sequencing (NGS), Sanger sequencing and immunohistochemistry (IHC) assays to assess LKB1 mutation and protein expression status in a set of FFPE patient specimens. Methods: For NGS, we used the Ion Torrent platform to sequence a 4.4kb region of interest (ROI) around LKB1. A combination of TorrentSuite and in-house tools were used for alignment, variant calling and prioritization of variants. Sanger sequencing was used to validate the presence and absence of variants with a frequency of at least 10% that were identified by NGS. An IHC assay was developed using a commercially available antibody. These assays were validated using a combination of cell lines, a genetically engineered mouse model and FFPE tissue for specificity, sensitivity, reproducibility and concordance. Results: 35 patient FFPE tissues from a variety of tumors including cervix, endometrium, pancreas, skin and lung were evaluated for LKB1 mutation status using NGS and IHC methods. About 96% of the coding region of LKB1 had sufficient sequencing coverage (read depth &amp;gt;500x) to reliably call low frequency somatic variants. Analytical validation using cell line dilutions demonstrated that our NGS assay is able to detect single base changes with frequencies as low as 2-5%, and small indels with frequencies as low as 5-10%. We identified 9 coding and splice site variants in our preliminary analysis of 18/35 of the FFPE specimens. We compared the variants in 7 of these FFPE samples to the variants identified in these samples by Sanger sequencing, and found 100% concordance between the variant calls, for NGS variants with frequencies greater than 20%. Sanger sequencing in the remaining FFPE samples is ongoing, and lower frequency NGS variants will be confirmed by other methods. We compared the validated NGS variants with the IHC results to examine the correlation of sequencing results with protein expression analysis. In our preliminary analysis of the first 18 FFPE samples, all of the samples with an IHC score of zero or one (IHC negative) had either a nonsense (n=1) or missense (n=4) variant in STK11. Conclusions: Our data suggest that accurate assessment of LKB1 status may require complementary methods including NGS and IHC methods. Citation Format: Kimberly Pelak, Jennifer Wright, Zhenyu Yan, Agus Darwanto, Weihua Liu, Peng Fang, Jin Li, Sabita Sankar, Chad Galderisi. Complementary analysis of LKB1/STK11 mutation and protein expression status using next-generation sequencing, Sanger sequencing and immunohistochemistry. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3509. doi:10.1158/1538-7445.AM2013-3509

De novo Transcriptome Sequencing Reveals a Considerable Bias in the Incidence of Simple Sequence Repeats towards the Downstream of ‘Pre-miRNAs’ of Black Pepper

Next generation sequencing has an advantageon transformational development of species with limited available sequence data as it helps to decode the genome and transcriptome. We carried out the de novo sequencing using illuminaHiSeq™ 2000 to generate the first leaf transcriptome of black pepper (Piper nigrum L.), an important spice variety native to South India and also grown in other tropical regions. Despite the economic and biochemical importance of pepper, a scientifically rigorous study at the molecular level is far from complete due to lack of sufficient sequence information and cytological complexity of its genome. The 55 million raw reads obtained, when assembled using Trinity program generated 2,23,386 contigs and 1,28,157 unigenes. Reports suggest that the repeat-rich genomic regions give rise to small non-coding functional RNAs. MicroRNAs (miRNAs) are the most abundant type of non-coding regulatory RNAs. In spite of the widespread research on miRNAs, little is known about the hair-pin precursors of miRNAs bearing Simple Sequence Repeats (SSRs). We used the array of transcripts generated, for the in silico prediction and detection of ‘43 pre-miRNA candidates bearing different types of SSR motifs’. The analysis identified 3913 different types of SSR motifs with an average of one SSR per 3.04 MB of thetranscriptome. About 0.033% of the transcriptome constituted ‘pre-miRNA candidates bearing SSRs’. The abundance, type and distribution of SSR motifs studied across the hair-pin miRNA precursors, showed a significant bias in the position of SSRs towards the downstream of predicted ‘pre-miRNA candidates’. The catalogue of transcripts identified, together with the demonstration of reliable existence of SSRs in the miRNA precursors, permits future opportunities for understanding the genetic mechanism of black pepper and likely functions of ‘tandem repeats’ in miRNAs.

Life in the dark: metagenomic evidence that a microbial slime community is driven by inorganic nitrogen metabolism

Beneath Australia's large, dry Nullarbor Plain lies an extensive underwater cave system, where dense microbial communities known as 'slime curtains' are found. These communities exist in isolation from photosynthetically derived carbon and are presumed to be chemoautotrophic. Earlier work found high levels of nitrite and nitrate in the cave waters and a high relative abundance of Nitrospirae in bacterial 16S rRNA clone libraries. This suggested that these communities may be supported by nitrite oxidation, however, details of the inorganic nitrogen cycling in these communities remained unclear. Here we report analysis of 16S rRNA amplicon and metagenomic sequence data from the Weebubbie cave slime curtain community. The microbial community is comprised of a diverse assortment of bacterial and archaeal genera, including an abundant population of Thaumarchaeota. Sufficient thaumarchaeotal sequence was recovered to enable a partial genome sequence to be assembled, which showed considerable synteny with the corresponding regions in the genome of the autotrophic ammonia oxidiser Nitrosopumilus maritimus SCM1. This partial genome sequence, contained regions with high sequence identity to the ammonia mono-oxygenase operon and carbon fixing 3-hydroxypropionate/4-hydroxybutyrate cycle genes of N. maritimus SCM1. Additionally, the community, as a whole, included genes encoding key enzymes for inorganic nitrogen transformations, including nitrification and denitrification. We propose that the Weebubbie slime curtain community represents a distinctive microbial ecosystem, in which primary productivity is due to the combined activity of archaeal ammonia-oxidisers and bacterial nitrite oxidisers.

Genomic patterns of introgression in rainbow and westslope cutthroat trout illuminated by overlapping paired‐end RAD sequencing

Rapid and inexpensive methods for genomewide single nucleotide polymorphism (SNP) discovery and genotyping are urgently needed for population management and conservation. In hybridized populations, genomic techniques that can identify and genotype thousands of species-diagnostic markers would allow precise estimates of population- and individual-level admixture as well as identification of 'super invasive' alleles, which show elevated rates of introgression above the genomewide background (likely due to natural selection). Techniques like restriction-site-associated DNA (RAD) sequencing can discover and genotype large numbers of SNPs, but they have been limited by the length of continuous sequence data they produce with Illumina short-read sequencing. We present a novel approach, overlapping paired-end RAD sequencing, to generate RAD contigs of >300-400 bp. These contigs provide sufficient flanking sequence for design of high-throughput SNP genotyping arrays and strict filtering to identify duplicate paralogous loci. We applied this approach in five populations of native westslope cutthroat trout that previously showed varying (low) levels of admixture from introduced rainbow trout (RBT). We produced 77 141 RAD contigs and used these data to filter and genotype 3180 previously identified species-diagnostic SNP loci. Our population-level and individual-level estimates of admixture were generally consistent with previous microsatellite-based estimates from the same individuals. However, we observed slightly lower admixture estimates from genomewide markers, which might result from natural selection against certain genome regions, different genomic locations for microsatellites vs. RAD-derived SNPs and/or sampling error from the small number of microsatellite loci (n = 7). We also identified candidate adaptive super invasive alleles from RBT that had excessively high admixture proportions in hybridized cutthroat trout populations.

Development of high‐density SNP genotyping arrays for white spruce (Picea glauca) and transferability to subtropical and nordic congeners

High-density SNP genotyping arrays can be designed for any species given sufficient sequence information of high quality. Two high-density SNP arrays relying on the Infinium iSelect technology (Illumina) were designed for use in the conifer white spruce (Picea glauca). One array contained 7338 segregating SNPs representative of 2814 genes of various molecular functional classes for main uses in genetic association and population genetics studies. The other one contained 9559 segregating SNPs representative of 9543 genes for main uses in population genetics, linkage mapping of the genome and genomic prediction. The SNPs assayed were discovered from various sources of gene resequencing data. SNPs predicted from high-quality sequences derived from genomic DNA reached a genotyping success rate of 64.7%. Nonsingleton in silico SNPs (i.e. a sequence polymorphism present in at least two reads) predicted from expressed sequenced tags obtained with the Roche 454 technology and Illumina GAII analyser resulted in a similar genotyping success rate of 71.6% when the deepest alignment was used and the most favourable SNP probe per gene was selected. A variable proportion of these SNPs was shared by other nordic and subtropical spruce species from North America and Europe. The number of shared SNPs was inversely proportional to phylogenetic divergence and standing genetic variation in the recipient species, but positively related to allele frequency in P. glauca natural populations. These validated SNP resources should open up new avenues for population genetics and comparative genetic mapping at a genomic scale in spruce species.