Library Insert Size Research Articles

Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis.

Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis.

Evaluating whole-genome sequencing quality metrics for enteric pathogen outbreaks.

BackgroundWhole genome sequencing (WGS) has gained increasing importance in responses to enteric bacterial outbreaks. Common analysis procedures for WGS, single nucleotide polymorphisms (SNPs) and genome assembly, are highly dependent upon WGS data quality.MethodsRaw, unprocessed WGS reads from Escherichia coli, Salmonella enterica, and Shigella sonnei outbreak clusters were characterized for four quality metrics: PHRED score, read length, library insert size, and ambiguous nucleotide composition. PHRED scores were strongly correlated with improved SNPs analysis results in E. coli and S. enterica clusters.ResultsAssembly quality showed only moderate correlations with PHRED scores and library insert size, and then only for Salmonella. To improve SNP analyses and assemblies, we compared seven read-healing pipelines to improve these four quality metrics and to see how well they improved SNP analysis and genome assembly. The most effective read healing pipelines for SNPs analysis incorporated quality-based trimming, fixed-width trimming, or both. The Lyve-SET SNPs pipeline showed a more marked improvement than the CFSAN SNP Pipeline, but the latter performed better on raw, unhealed reads. For genome assembly, SPAdes enabled significant improvements in healed E. coli reads only, while Skesa yielded no significant improvements on healed reads.ConclusionsPHRED scores will continue to be a crucial quality metric albeit not of equal impact across all types of analyses for all enteric bacteria. While trimming-based read healing performed well for SNPs analyses, different read healing approaches are likely needed for genome assembly or other, emerging WGS analysis methodologies.

A study of transposable element-associated structural variations (TASVs) using a de novo-assembled Korean genome

Advances in next-generation sequencing (NGS) technology have made personal genome sequencing possible, and indeed, many individual human genomes have now been sequenced. Comparisons of these individual genomes have revealed substantial genomic differences between human populations as well as between individuals from closely related ethnic groups. Transposable elements (TEs) are known to be one of the major sources of these variations and act through various mechanisms, including de novo insertion, insertion-mediated deletion, and TE–TE recombination-mediated deletion. In this study, we carried out de novo whole-genome sequencing of one Korean individual (KPGP9) via multiple insert-size libraries. The de novo whole-genome assembly resulted in 31,305 scaffolds with a scaffold N50 size of 13.23 Mb. Furthermore, through computational data analysis and experimental verification, we revealed that 182 TE-associated structural variation (TASV) insertions and 89 TASV deletions contributed 64,232 bp in sequence gain and 82,772 bp in sequence loss, respectively, in the KPGP9 genome relative to the hg19 reference genome. We also verified structural differences associated with TASVs by comparative analysis with TASVs in recent genomes (AK1 and TCGA genomes) and reported their details. Here, we constructed a new Korean de novo whole-genome assembly and provide the first study, to our knowledge, focused on the identification of TASVs in an individual Korean genome. Our findings again highlight the role of TEs as a major driver of structural variations in human individual genomes.

The sequence and de novo assembly of the wild yak genome

Vulnerable populations of wild yak (Bos mutus), the wild ancestral species of domestic yak, survive in extremely cold, harsh and oxygen-poor regions of the Qinghai-Tibetan Plateau (QTP) and adjacent high-altitude regions. In this study, we sequenced and assembled its genome de novo. In total, six different insert-size libraries were sequenced, and 662 Gb of clean data were generated. The assembled wild yak genome is 2.83 Gb in length, with an N50 contig size of 63.2 kb and a scaffold size of 16.3 Mb. BUSCO assessment indicated that 93.8% of the highly conserved mammal genes were completely present in the genome assembly. Annotation of the wild yak genome assembly identified 1.41 Gb (49.65%) of repetitive sequences and a total of 22,910 protein-coding genes, including 20,660 (90.18%) annotated with functional terms. This first construction of the wild yak genome provides a variable genetic resource that will facilitate further study of the genetic diversity of bovine species and accelerate yak breeding efforts.

Advantages of Single-Stranded DNA Over Double-Stranded DNA Library Preparation for Capturing Cell-Free Tumor DNA in Plasma.

Single-stranded DNA (ssDNA) libraries have been shown to enrich shorter and more degraded DNA fragments than double-stranded DNA (dsDNA) libraries. In this study, we evaluated whether ssDNA libraries captured more circulating tumor DNA (ctDNA) in plasma cell-free DNA (cfDNA). We prepared dsDNA, ssDNA and pure-ssDNA (capture the preexisting ssDNA) libraries using ten plasma cfDNA samples. After low-pass whole genome sequencing, we calculated a duplicate rate to estimate library complexity and compared the library insert sizes between the different library methods. Finally, we estimated the ctDNA content and plasma genomic abnormality (PGA) score, an indicator of ctDNA burden. 27 libraries were prepared and sequenced from the ten cfDNA samples. The duplicate rate in the ssDNA and pure-ssDNA libraries was significantly lower than in the dsDNA libraries (p < 0.001 and p < 0.01, respectively). ctDNA content and PGA scores were consistently higher in the ssDNA and pure-ssDNA libraries than in the matched dsDNA libraries (p < 0.005). The higher ctDNA content in ssDNA libraries was associated with smaller library insert size. ssDNA libraries preserve more diversity and capture more ctDNA than dsDNA libraries. The ssDNA library method is preferred when performing genomic analysis of ctDNA.

Abstract 3536: Advantages of ssDNA over dsDNA library preparation for capturing cell-free tumor DNA in plasma

Abstract Single-stranded DNA (ssDNA) sequencing library method has shown to enrich shorter and degraded DNA fragments than double-stranded DNA (dsDNA) library. The goal of this study is to determine whether ssDNA libraries enriches more circulating tumor DNA (ctDNA) in plasma cell-free DNA (cfDNA). We used 2ng cfDNA to prepare dsDNA libraries and ssDNA libraries. We also prepare pure-ssDNA libraries by skipping the DNA denaturation step to capture the pre-existing ssDNA only. To calculate ctDNA content, we mapped fastq files to human genome and binned the mapped reads into 1 Mb genomic windows. We normalized the read counts using two unrelated healthy controls. The resulting ratios were transformed with log2 and adjusted for GC content. The fully normalized log2 ratios were subjected to segmentation using the copy number analysis method. After the segmentation, we selected the most significantly deleted genomic region with segment size &amp;gt;20 Mb in each library pair and calculated ctDNA content using formula: 100 × (1-2log2 ratio). We also calculated the plasma genomic abnormality (PGA) score (a composite score algorithm to reflect overall ctDNA burden in peripheral blood) to estimate ctDNA burden. Overall, we prepared and sequenced a total of 27 libraries including ten dsDNA libraries, ten ssDNA libraries and seven pure-ssDNA libraries, using ten cfDNAs from cancer patients. We received an average of 24264998 raw read sequences and 19495014 mappable reads per sequencing library. Duplicate rate in the ssDNA libraries (mean=0.057%, range=0.046-0.083%) and pure-ssDNA libraries (mean=0.06%, range= 0.054-0.065%) was significantly lower than in dsDNA libraries (mean=0.2%, range=0.14-0.249%) (p&amp;lt;0.001 and p&amp;lt;0.01, respectively), indicating that ssDNA-based library preparation method may better preserve the diversity and complexity of ctDNAs. Library insert size analysis showed that ssDNA libraries were on average 14bp shorter (range from 6 bp to 21 bp) than their matched dsDNA libraries. We also found the Ampure XP beads ratio influenced the library size. We observed consistently higher ctDNA content in ssDNA libraries than in matched dsDNA libraries (p&amp;lt;0.0005). Comparing ssDNA libraries (mean ctDNA content=34%) to their matched dsDNA libraries (mean ctDNA content=30.7%), the increased ctDNA content ranged from 1.3% to 6.4% (mean=3.3%). ctDNA content was also significantly higher in pure-ssDNA libraries than in their matched dsDNA libraries (p&amp;lt;0.005). The increased ctDNA content ranged from 1.0% to 6.9% (mean=3.6%). The PGA scores were always higher in the ssDNA libraries (p&amp;lt;0.0001) or pure-ssDNA libraries (p&amp;lt;0.005) compared to the matched dsDNA method. Our results demonstrate that ssDNA libraries enrich more ctDNA than dsDNA libraries do. Pre-existing ssDNA in plasma is abundant and provides sufficient resource for genomic analysis of ctDNA. Citation Format: Jing Zhu, Jingyong Huang, Peng Zhang, Manish Kohli, Chiang-Ching Huang, Liang Wang. Advantages of ssDNA over dsDNA library preparation for capturing cell-free tumor DNA in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3536.

The sequence and de novo assembly of hog deer genome

Hog deer (Axis porcinus) is a small deer species in family Cervidae and has been undergoing a serious and global decline during the past decades. Chengdu Zoo currently holds a captive population of hog deer with sufficient genetic diversity in China. We sequenced and de novo assembled its genome sequence in the present study. A total of six different insert-size libraries were sequenced and generated 395 Gb of clean data in total. With aid of the linked reads of 10X Genomics, genome sequence was assembled to 2.72 Gb in length (contig N50, 66.04 Kb; scaffold N50, 20.55 Mb), in which 94.5% of expected genes were detected. We comprehensively annotated 22,473 protein-coding genes, 37,019 tRNAs, and 1,058 Mb repeated sequences. The newly generated reference genome is expected to significantly contribute to comparative analysis of genome biology and evolution within family Cervidae.

Genome sequencing and conservation genomics in the Scandinavian wolverine population.

Genetic approaches have proved valuable to the study and conservation of endangered populations, especially for monitoring programs, and there is potential for further developments in this direction by extending analyses to the genomic level. We assembled the genome of the wolverine (Gulo gulo), a mustelid that in Scandinavia has recently recovered from a significant population decline, and obtained a 2.42Gb draft sequence representing >85% of the genome and including >21,000protein-coding genes. We then performed whole-genome resequencing of 10Scandinavian wolverines for population genomic and demographic analyses. Genetic diversity was among the lowest detected in a red-listed population (mean genome-wide nucleotide diversity of 0.05%). Results of the demographic analyses indicated a long-term decline of the effective population size (Ne ) from 10,000well before the last glaciation to <500 after this period. Current Ne appeared even lower. The genome-wide FIS level was 0.089 (possibly signaling inbreeding), but this effect was not observed when analyzing a set of highly variable SNP markers, illustrating that such markers can give a biased picture of the overall character of genetic diversity. We found significant population structure, which has implications for population connectivity and conservation. We used an integrated microfluidic circuit chip technology to develop an SNP-array consisting of 96 highly informative markers that, together with a multiplex pre-amplification step, was successfully applied to low-quality DNA from scat samples. Our findings will inform management, conservation, and genetic monitoring of wolverines and serve as a genomic roadmap that can be applied to other endangered species. The approach used here can be generally utilized in other systems, but we acknowledge the trade-off between investing in genomic resources and direct conservation actions.

Draft genome of the gayal, Bos frontalis

Gayal (Bos frontalis), also known as mithan or mithun, is a large endangered semi-domesticated bovine that has a limited geographical distribution in the hill-forests of China, Northeast India, Bangladesh, Myanmar, and Bhutan. Many questions about the gayal such as its origin, population history, and genetic basis of local adaptation remain largely unresolved. De novo sequencing and assembly of the whole gayal genome provides an opportunity to address these issues. We report a high-depth sequencing, de novo assembly, and annotation of a female Chinese gayal genome. Based on the Illumina genomic sequencing platform, we have generated 350.38 Gb of raw data from 16 different insert-size libraries. A total of 276.86 Gb of clean data is retained after quality control. The assembled genome is about 2.85 Gb with scaffold and contig N50 sizes of 2.74 Mb and 14.41 kb, respectively. Repetitive elements account for 48.13% of the genome. Gene annotation has yielded 26 667 protein-coding genes, of which 97.18% have been functionally annotated. BUSCO assessment shows that our assembly captures 93% (3183 of 4104) of the core eukaryotic genes and 83.1% of vertebrate universal single-copy orthologs. We provide the first comprehensive de novo genome of the gayal. This genetic resource is integral for investigating the origin of the gayal and performing comparative genomic studies to improve understanding of the speciation and divergence of bovine species. The assembled genome could be used as reference in future population genetic studies of gayal.

Abstract 5410: Optimization and evaluation of SMART-Seq v4 kit for low input RNA sequencing

Abstract Recent advances in library preparation methodology from limiting amounts of total RNA have facilitated the characterization of rare cell-types in various biological systems. The SMART-Seq v4 Ultra Low Input RNA Kit incorporates a number of workflow improvements, including fewer purifications to increase yield, a locked nucleic acid (LNA) template-switching oligo to enhance stability, and an improved polymerase designed to reduce amplification bias of GC-rich regions. Certain workflow challenges remain however, including variability in library insert size, which can lead to less predictable sequencing results. We have modified the SMART-Seq v4 method to incorporate library size selection between 250-650 bp, and have characterized analytical performance of the modified procedure. A total of 63 libraries were generated using 10 pg - 10 ng of Universal Human Reference RNA (UHRR), Human Brain Reference RNA (HBRR), and total RNA isolated from multiple human tissues, including lung, colon, spleen, adrenal tumor, heart, and lung tumor. To facilitate the comparison between libraries, we normalized library read counts by down-sampling to 24 million reads prior to further processing with an internally-developed sequence analysis pipeline (RNAv9_rsem). The values for ERCC Limit of Detection ranged between 3-50 copies with a mean of 22 copies, which confirmed the high absolute sensitivity of SMART-seq v4 workflow. The total number of genes detected (RPKM, or reads per kilobase per million mapped &amp;gt;=3) varied as a function of the amount of input RNA, with as many as 19,500 genes detected. Gene detection was highly consistent across replicates utilizing 100 pg-10 ng input RNA, with a precipitous decline in genes detected using 10 pg of RNA input. This is due to less reliable detection of low abundance genes, which in turn leads to increased discordance in detected genes between replicates, possibly due to increased sampling error. Nonetheless, when genes are commonly detected, their normalized read counts are highly correlated at any given RNA input, with correlation coefficients (r) ranging from 0.977 to 0.993. Compared to previous versions of the SMARTer method, the modified SMART-Seq v4 procedure implemented at Q2 Solutions - EA Genomics significantly improved the percentage of reads aligned to the transcriptome, as well as the total number of genes detected, all with reduced technical variability. Our study demonstrates an improved strategy for expression profiling via RNA sequencing from limiting amounts of RNA. Citation Format: Dan Su, Thomas Halsey, Martin Buchkovich, Jason G. Powers, Steven Abbott, Jenny Shu, John Pufky, Michell Chang, Victor Weigman, Patrick Hurban. Optimization and evaluation of SMART-Seq v4 kit for low input RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5410. doi:10.1158/1538-7445.AM2017-5410

Improved genome sequencing using an engineered transposase

BackgroundNext-generation sequencing (NGS) has transformed genomic research by reducing turnaround time and cost. However, no major breakthrough has been made in the upstream library preparation methods until the transposase-based Nextera method was invented. Nextera combines DNA fragmentation and barcoding in a single tube reaction and therefore enables a very fast workflow to sequencing-ready DNA libraries within a couple of hours. When compared to the traditional ligation-based methods, transposed-based Nextera has a slight insertion bias.ResultsHere we present the discovery of a mutant transposase (Tn5-059) with a lowered GC insertion bias through protein engineering. We demonstrate Tn5-059 reduces AT dropout and increases uniformity of genome coverage in both bacterial genomes and human genome. We also observe higher library diversity generated by Tn5-059 when compared to Nextera v2 for human exomes, which leads to less sequencing and lower cost per genome. In addition, when used for human exomes, Tn5-059 delivers consistent library insert size over a range of input DNA, allowing up to a tenfold variance from the 50 ng input recommendation.ConclusionsEnhanced DNA input tolerance of Tn5-059 can translate to flexibility and robustness of workflow. DNA input tolerance together with superior uniformity of coverage and lower AT dropouts extend the applications of transposase based library preps. We discuss possible mechanisms of improvements in Tn5-059, and potential advantages of using the new mutant in varieties of applications including microbiome sequencing and chromatin profiling.

A Step-by-Step Guide to Assemble a Reptilian Genome.

Multiple technologies and software are now available facilitating the de novo sequencing and assembly of any vertebrate genome. Yet the quality of most available sequenced genomes is substantially poorer than that of the golden standard in the field: the human genome. Here, we present a step-by-step protocol for the successful sequencing and assembly of a high-quality snake genome that can be applied to any other reptilian or avian species. We combine the great sequencing depth and accuracy of short reads with the use of different insert size libraries for extended scaffolding followed by optical mapping. We show that this procedure improved the corn snake scaffold N50 from 3.7kbp to 1.4Mbp, currently making it one of the snake genomes with the longest scaffolds. Short guidelines are also given on the extraction of long DNA molecules from reptilian blood and the necessary modifications in DNA extraction protocols. This chapter is accompanied by a website ( www.reptilomics.org/stepbystep.html ), where we provide links to the suggested software, examples of input and output files, and running parameters.

Optimized Illumina PCR-free library preparation for bacterial whole genome sequencing and analysis of factors influencing de novo assembly

BackgroundNext-generation sequencing (NGS) technology has paved the way for rapid and cost-efficient de novo sequencing of bacterial genomes. In particular, the introduction of PCR-free library preparation procedures (LPPs) lead to major improvements as PCR bias is largely reduced. However, in order to facilitate the assembly of Illumina paired-end sequence data and to enhance assembly performance, an increase of insert sizes to facilitate the repeat bridging and resolution capabilities of current state of the art assembly tools is needed. In addition, information concerning the relationships between genomic GC content, library insert size and sequencing quality as well as the influence of library insert size, read length and sequencing depth on assembly performance would be helpful to specifically target sequencing projects.ResultsOptimized DNA fragmentation settings and fine-tuned resuspension buffer to bead buffer ratios during fragment size selection were integrated in the Illumina TruSeq® DNA PCR-free LPP in order to produce sequencing libraries varying in average insert size for bacterial genomes within a range of 35.4–73.0 % GC content. The modified protocol consumes only half of the reagents per sample, thus doubling the number of preparations possible with a kit. Examination of different libraries revealed that sequencing quality decreases with increased genomic GC content and with larger insert sizes. The estimation of assembly performance using assembly metrics like corrected NG50 and NGA50 showed that libraries with larger insert sizes can result in substantial assembly improvements as long as appropriate assembly tools are chosen. However, such improvements seem to be limited to genomes with a low to medium GC content. A positive trend between read length and assembly performance was observed while sequencing depth is less important, provided a minimum coverage is reached.ConclusionsBased on the optimized protocol developed, sequencing libraries with flexible insert sizes and lower reagent costs can be generated. Furthermore, increased knowledge about the interplay of sequencing quality, insert size, genomic GC content, read length, sequencing depth and the assembler used will help molecular biologists to set up an optimal experimental and analytical framework with respect to Illumina next-generation sequencing of bacterial genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2072-9) contains supplementary material, which is available to authorized users.

Optimizing and benchmarking de novo transcriptome sequencing: from library preparation to assembly evaluation.

BackgroundRNA-seq enables gene expression profiling in selected spatiotemporal windows and yields massive sequence information with relatively low cost and time investment, even for non-model species. However, there remains a large room for optimizing its workflow, in order to take full advantage of continuously developing sequencing capacity.MethodTranscriptome sequencing for three embryonic stages of Madagascar ground gecko (Paroedura picta) was performed with the Illumina platform. The output reads were assembled de novo for reconstructing transcript sequences. In order to evaluate the completeness of transcriptome assemblies, we prepared a reference gene set consisting of vertebrate one-to-one orthologs.ResultTo take advantage of increased read length of >150 nt, we demonstrated shortened RNA fragmentation time, which resulted in a dramatic shift of insert size distribution. To evaluate products of multiple de novo assembly runs incorporating reads with different RNA sources, read lengths, and insert sizes, we introduce a new reference gene set, core vertebrate genes (CVG), consisting of 233 genes that are shared as one-to-one orthologs by all vertebrate genomes examined (29 species)., The completeness assessment performed by the computational pipelines CEGMA and BUSCO referring to CVG, demonstrated higher accuracy and resolution than with the gene set previously established for this purpose. As a result of the assessment with CVG, we have derived the most comprehensive transcript sequence set of the Madagascar ground gecko by means of assembling individual libraries followed by clustering the assembled sequences based on their overall similarities.ConclusionOur results provide several insights into optimizing de novo RNA-seq workflow, including the coordination between library insert size and read length, which manifested in improved connectivity of assemblies. The approach and assembly assessment with CVG demonstrated here would be applicable to transcriptome analysis of other species as well as whole genome analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2007-1) contains supplementary material, which is available to authorized users.

Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community.

BackgroundThe rapid development of sequencing technologies has provided access to environments that were either once thought inhospitable to life altogether or that contain too few cells to be analyzed using genomics approaches. While 16S rRNA gene microbial community sequencing has revolutionized our understanding of community composition and diversity over time and space, it only provides a crude estimate of microbial functional and metabolic potential. Alternatively, shotgun metagenomics allows comprehensive sampling of all genetic material in an environment, without any underlying primer biases. Until recently, one of the major bottlenecks of shotgun metagenomics has been the requirement for large initial DNA template quantities during library preparation.ResultsHere, we investigate the effects of varying template concentrations across three low biomass library preparation protocols on their ability to accurately reconstruct a mock microbial community of known composition. We analyze the effects of input DNA quantity and library preparation method on library insert size, GC content, community composition, assembly quality and metagenomic binning. We found that library preparation method and the amount of starting material had significant impacts on the mock community metagenomes. In particular, GC content shifted towards more GC rich sequences at the lower input quantities regardless of library prep method, the number of low quality reads that could not be mapped to the reference genomes increased with decreasing input quantities, and the different library preparation methods had an impact on overall metagenomic community composition.ConclusionsThis benchmark study provides recommendations for library creation of representative and minimally biased metagenome shotgun sequencing, enabling insights into functional attributes of low biomass ecosystem microbial communities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2063-6) contains supplementary material, which is available to authorized users.

Population-based structural variation discovery with Hydra-Multi.

Summary: Current strategies for SNP and INDEL discovery incorporate sequence alignments from multiple individuals to maximize sensitivity and specificity. It is widely accepted that this approach also improves structural variant (SV) detection. However, multisample SV analysis has been stymied by the fundamental difficulties of SV calling, e.g. library insert size variability, SV alignment signal integration and detecting long-range genomic rearrangements involving disjoint loci. Extant tools suffer from poor scalability, which limits the number of genomes that can be co-analyzed and complicates analysis workflows. We have developed an approach that enables multisample SV analysis in hundreds to thousands of human genomes using commodity hardware. Here, we describe Hydra-Multi and measure its accuracy, speed and scalability using publicly available datasets provided by The 1000 Genomes Project and by The Cancer Genome Atlas (TCGA).Availability and implementation: Hydra-Multi is written in C++ and is freely available at https://github.com/arq5x/Hydra.Contact: aaronquinlan@gmail.com or ihall@genome.wustl.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Comparative genomic data of the Avian Phylogenomics Project.

BackgroundThe evolutionary relationships of modern birds are among the most challenging to understand in systematic biology and have been debated for centuries. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae and two of the five Palaeognathae orders, and used the genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomics analyses (Jarvis et al. in press; Zhang et al. in press). Here we release assemblies and datasets associated with the comparative genome analyses, which include 38 newly sequenced avian genomes plus previously released or simultaneously released genomes of Chicken, Zebra finch, Turkey, Pigeon, Peregrine falcon, Duck, Budgerigar, Adelie penguin, Emperor penguin and the Medium Ground Finch. We hope that this resource will serve future efforts in phylogenomics and comparative genomics.FindingsThe 38 bird genomes were sequenced using the Illumina HiSeq 2000 platform and assembled using a whole genome shotgun strategy. The 48 genomes were categorized into two groups according to the N50 scaffold size of the assemblies: a high depth group comprising 23 species sequenced at high coverage (>50X) with multiple insert size libraries resulting in N50 scaffold sizes greater than 1 Mb (except the White-throated Tinamou and Bald Eagle); and a low depth group comprising 25 species sequenced at a low coverage (~30X) with two insert size libraries resulting in an average N50 scaffold size of about 50 kb. Repetitive elements comprised 4%-22% of the bird genomes. The assembled scaffolds allowed the homology-based annotation of 13,000 ~ 17000 protein coding genes in each avian genome relative to chicken, zebra finch and human, as well as comparative and sequence conservation analyses.ConclusionsHere we release full genome assemblies of 38 newly sequenced avian species, link genome assembly downloads for the 7 of the remaining 10 species, and provide a guideline of genomic data that has been generated and used in our Avian Phylogenomics Project. To the best of our knowledge, the Avian Phylogenomics Project is the biggest vertebrate comparative genomics project to date. The genomic data presented here is expected to accelerate further analyses in many fields, including phylogenetics, comparative genomics, evolution, neurobiology, development biology, and other related areas.Electronic supplementary materialThe online version of this article (doi:10.1186/2047-217X-3-26) contains supplementary material, which is available to authorized users.

Next-generation sequencing of custom amplicons to improve coverage of HaloPlex multigene panels.

Next-generation sequencing (NGS) of multigene panels performed for genetic clinical diagnostics requires 100% coverage of all targeted genes. In the genetic diagnostics laboratory, coverage gaps are typically filled with Sanger sequencing after NGS data are collected and analyzed. Libraries prepared using the hybridization-based custom capture HaloPlex method are covered at ~98% and include gaps in coverage because of the location of the restriction enzyme sites used for fragmentation and differences in the designed and actual library insert size. We describe a method for improving the coverage of HaloPlex libraries by generating a set of amplicons spanning known low-coverage regions that are pooled, indexed by sample, and sequenced together with the HaloPlex libraries. This approach reduces the number of post-NGS Sanger sequencing reactions required and complements any NGS library preparation method when complete gene coverage is necessary.

Genomic Takeover by Transposable Elements in the Strawberry Poison Frog.

We sequenced the genome of the strawberry poison frog, Oophaga pumilio, at a depth of 127.5× using variable insert size libraries. The total genome size is estimated to be 6.76 Gb, of which 4.76 Gb are from high copy number repetitive elements with low differentiation across copies. These repeats encompass DNA transposons, RNA transposons, and LTR retrotransposons, including at least 0.4 and 1.0 Gb of Mariner/Tc1 and Gypsy elements, respectively. Expression data indicate high levels of gypsy and Mariner/Tc1 expression in ova of O. pumilio compared with Xenopus laevis. We further observe phylogenetic evidence for horizontal transfer (HT) of Mariner elements, possibly between fish and frogs. The elements affected by HT are present in high copy number and are highly expressed, suggesting ongoing proliferation after HT. Our results suggest that the large amphibian genome sizes, at least partially, can be explained by a process of repeated invasion of new transposable elements that are not yet suppressed in the germline. We also find changes in the spliceosome that we hypothesize are related to permissiveness of O. pumilio to increases in intron length due to transposon proliferation. Finally, we identify the complement of ion channels in the first genomic sequenced poison frog and discuss its relation to the evolution of autoresistance to toxins sequestered in the skin.

BESST--efficient scaffolding of large fragmented assemblies.

BackgroundThe use of short reads from High Throughput Sequencing (HTS) techniques is now commonplace in de novo assembly. Yet, obtaining contiguous assemblies from short reads is challenging, thus making scaffolding an important step in the assembly pipeline. Different algorithms have been proposed but many of them use the number of read pairs supporting a linking of two contigs as an indicator of reliability. This reasoning is intuitive, but fails to account for variation in link count due to contig features.We have also noted that published scaffolders are only evaluated on small datasets using output from only one assembler. Two issues arise from this. Firstly, some of the available tools are not well suited for complex genomes. Secondly, these evaluations provide little support for inferring a software’s general performance.ResultsWe propose a new algorithm, implemented in a tool called BESST, which can scaffold genomes of all sizes and complexities and was used to scaffold the genome of P. abies (20 Gbp). We performed a comprehensive comparison of BESST against the most popular stand-alone scaffolders on a large variety of datasets. Our results confirm that some of the popular scaffolders are not practical to run on complex datasets. Furthermore, no single stand-alone scaffolder outperforms the others on all datasets. However, BESST fares favorably to the other tested scaffolders on GAGE datasets and, moreover, outperforms the other methods when library insert size distribution is wide.ConclusionWe conclude from our results that information sources other than the quantity of links, as is commonly used, can provide useful information about genome structure when scaffolding.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2105-15-281) contains supplementary material, which is available to authorized users.