Museum genomics: low-cost and high-accuracy genetic data from historical specimens

Natural history collections are unparalleled repositories of geographical and temporal variation in faunal conditions. Molecular studies offer an opportunity to uncover much of this variation; however, genetic studies of historical museum specimens typically rely on extracting highly degraded and chemically modified DNA samples from skins, skulls or other dried samples. Despite this limitation, obtaining short fragments of DNA sequences using traditional PCR amplification of DNA has been the primary method for genetic study of historical specimens. Few laboratories have succeeded in obtaining genome-scale sequences from historical specimens and then only with considerable effort and cost. Here, we describe a low-cost approach using high-throughput next-generation sequencing to obtain reliable genome-scale sequence data from a traditionally preserved mammal skin and skull using a simple extraction protocol. We show that single-nucleotide polymorphisms (SNPs) from the genome sequences obtained independently from the skin and from the skull are highly repeatable compared to a reference genome.     

A Microfluidic DNA Library Preparation Platform for Next-Generation Sequencing

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.     

Protein Domain Analysis of Genomic Sequence Data Reveals Regulation of LRR Related Domains in Plant Transpiration in Ficus

Predicting protein domains is essential for understanding a protein’s function at the molecular level. However, up till now, there has been no direct and straightforward method for predicting protein domains in species without a reference genome sequence. In this study, we developed a functionality with a set of programs that can predict protein domains directly from genomic sequence data without a reference genome. Using whole genome sequence data, the programming functionality mainly comprised DNA assembly in combination with next-generation sequencing (NGS) assembly methods and traditional methods, peptide prediction and protein domain prediction. The proposed new functionality avoids problems associated with de novo assembly due to micro reads and small single repeats. Furthermore, we applied our functionality for the prediction of leucine rich repeat (LRR) domains in four species of Ficus with no reference genome, based on NGS genomic data. We found that the LRRNT_2 and LRR_8 domains are related to plant transpiration efficiency, as indicated by the stomata index, in the four species of Ficus. The programming functionality established in this study provides new insights for protein domain prediction, which is particularly timely in the current age of NGS data expansion.     

Identification of Optimum Sequencing Depth Especially for De Novo Genome Assembly of Small Genomes Using Next Generation Sequencing Data

Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6–40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.     

NSIT: Novel Sequence Identification Tool

Novel sequences are DNA sequences present in an individual''s genome but absent in the human reference assembly. They are predicted to be biologically important, both individual and population specific, and consistent with the known human migration paths. Recent works have shown that an average person harbors 2–5 Mb of such sequences and estimated that the human pan-genome contains as high as 19–40 Mb of novel sequences. To identify them in a de novo genome assembly, some existing sequence aligners have been used but no computational method has been specifically proposed for this task. In this work, we developed NSIT (Novel Sequence Identification Tool), a software that can accurately and efficiently identify novel sequences in an individual''s de novo whole genome assembly. We identified and characterized 1.1 Mb, 1.2 Mb, and 1.0 Mb of novel sequences in NA18507 (African), YH (Asian), and NA12878 (European) de novo genome assemblies, respectively. Our results show very high concordance with the previous work using the respective reference assembly. In addition, our results using the latest human reference assembly suggest that the amount of novel sequences per individual may not be as high as previously reported. We additionally developed a graphical viewer for comparisons of novel sequence contents. The viewer also helped in identifying sequence contamination; we found 130 kb of Epstein-Barr virus sequence in the previously published NA18507 novel sequences as well as 287 kb of zebrafish repeats in NA12878 de novo assembly. NSIT requires 2GB of RAM and 1.5–2 hrs on a commodity desktop. The program is applicable to input assemblies with varying contig/scaffold sizes, ranging from 100 bp to as high as 50 Mb. It works in both 32-bit and 64-bit systems and outperforms, by large margins, other fast sequence aligners previously applied to this task. To our knowledge, NSIT is the first software designed specifically for novel sequence identification in a de novo human genome assembly.     

Sequencing Degraded DNA from Non-Destructively Sampled Museum Specimens for RAD-Tagging and Low-Coverage Shotgun Phylogenetics

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37±0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.     

Paired-End Sequencing of Long-Range DNA Fragments for De Novo Assembly of Large,Complex Mammalian Genomes by Direct Intra-Molecule Ligation

Background

The relatively short read lengths from next generation sequencing (NGS) technologies still pose a challenge for de novo assembly of complex mammal genomes. One important solution is to use paired-end (PE) sequence information experimentally obtained from long-range DNA fragments (>1 kb). Here, we characterize and extend a long-range PE library construction method based on direct intra-molecule ligation (or molecular linker-free circularization) for NGS.

Results

We found that the method performs stably for PE sequencing of 2- to 5- kb DNA fragments, and can be extended to 10–20 kb (and even in extremes, up to ∼35 kb). We also characterized the impact of low quality input DNA on the method, and develop a whole-genome amplification (WGA) based protocol using limited input DNA (<1 µg). Using this PE dataset, we accurately assembled the YanHuang (YH) genome, the first sequenced Asian genome, into a scaffold N50 size of >2 Mb, which is over100-times greater than the initial size produced with only small insert PE reads(17 kb). In addition, we mapped two 7- to 8- kb insertions in the YH genome using the larger insert sizes of the long-range PE data.

Conclusions

In conclusion, we demonstrate here the effectiveness of this long-range PE sequencing method and its use for the de novo assembly of a large, complex genome using NGS short reads.     

Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC) clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum). Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete.     

Long-Range Genomic Enrichment,Sequencing, and Assembly to Determine Unknown Sequences Flanking a Known microRNA

Conserved plant microRNAs (miRNAs) modulate important biological processes but little is known about conserved cis-regulatory elements (CREs) surrounding MIRNA genes. We developed a solution-based targeted genomic enrichment methodology to capture, enrich, and sequence flanking genomic regions surrounding conserved MIRNA genes with a locked-nucleic acid (LNA)-modified, biotinylated probe complementary to the mature miRNA sequence. Genomic DNA bound by the probe is captured by streptavidin-coated magnetic beads, amplified, sequenced and assembled de novo to obtain genomic DNA sequences flanking MIRNA locus of interest. We demonstrate the sensitivity and specificity of this enrichment methodology in Arabidopsis thaliana to enrich targeted regions spanning 10–20 kb surrounding known MIR166 and MIR165 loci. Assembly of the sequencing reads successfully recovered all targeted loci. While further optimization for larger, more complex genomes is needed, this method may enable determination of flanking genomic DNA sequence surrounding a known core (like a conserved mature miRNA) from multiple species that currently don''t have a full genome assembly available.     

Draft Sequences of the Radish (Raphanus sativus L.) Genome

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.     

Illumina TruSeq Synthetic Long-Reads Empower De Novo Assembly and Resolve Complex,Highly-Repetitive Transposable Elements

High-throughput DNA sequencing technologies have revolutionized genomic analysis, including the de novo assembly of whole genomes. Nevertheless, assembly of complex genomes remains challenging, in part due to the presence of dispersed repeats which introduce ambiguity during genome reconstruction. Transposable elements (TEs) can be particularly problematic, especially for TE families exhibiting high sequence identity, high copy number, or complex genomic arrangements. While TEs strongly affect genome function and evolution, most current de novo assembly approaches cannot resolve long, identical, and abundant families of TEs. Here, we applied a novel Illumina technology called TruSeq synthetic long-reads, which are generated through highly-parallel library preparation and local assembly of short read data and which achieve lengths of 1.5–18.5 Kbp with an extremely low error rate (0.03% per base). To test the utility of this technology, we sequenced and assembled the genome of the model organism Drosophila melanogaster (reference genome strain y; cn, bw, sp) achieving an N50 contig size of 69.7 Kbp and covering 96.9% of the euchromatic chromosome arms of the current reference genome. TruSeq synthetic long-read technology enables placement of individual TE copies in their proper genomic locations as well as accurate reconstruction of TE sequences. We entirely recovered and accurately placed 4,229 (77.8%) of the 5,434 annotated transposable elements with perfect identity to the current reference genome. As TEs are ubiquitous features of genomes of many species, TruSeq synthetic long-reads, and likely other methods that generate long-reads, offer a powerful approach to improve de novo assemblies of whole genomes.     

Construction of Pseudomolecule Sequences of the aus Rice Cultivar Kasalath for Comparative Genomics of Asian Cultivated Rice

Having a deep genetic structure evolved during its domestication and adaptation, the Asian cultivated rice (Oryza sativa) displays considerable physiological and morphological variations. Here, we describe deep whole-genome sequencing of the aus rice cultivar Kasalath by using the advanced next-generation sequencing (NGS) technologies to gain a better understanding of the sequence and structural changes among highly differentiated cultivars. The de novo assembled Kasalath sequences represented 91.1% (330.55 Mb) of the genome and contained 35 139 expressed loci annotated by RNA-Seq analysis. We detected 2 787 250 single-nucleotide polymorphisms (SNPs) and 7393 large insertion/deletion (indel) sites (>100 bp) between Kasalath and Nipponbare, and 2 216 251 SNPs and 3780 large indels between Kasalath and 93-11. Extensive comparison of the gene contents among these cultivars revealed similar rates of gene gain and loss. We detected at least 7.39 Mb of inserted sequences and 40.75 Mb of unmapped sequences in the Kasalath genome in comparison with the Nipponbare reference genome. Mapping of the publicly available NGS short reads from 50 rice accessions proved the necessity and the value of using the Kasalath whole-genome sequence as an additional reference to capture the sequence polymorphisms that cannot be discovered by using the Nipponbare sequence alone.     

Complete mitochondrial genomes of eleven extinct or possibly extinct bird species

Natural history museum collections represent a vast source of ancient and historical DNA samples from extinct taxa that can be utilized by high‐throughput sequencing tools to reveal novel genetic and phylogenetic information about them. Here, we report on the successful sequencing of complete mitochondrial genome sequences (mitogenomes) from eleven extinct bird species, using de novo assembly of short sequences derived from toepad samples of degraded DNA from museum specimens. For two species (the Passenger Pigeon Ectopistes migratorius and the South Island Piopio Turnagra capensis), whole mitogenomes were already available from recent studies, whereas for five others (the Great Auk Pinguinis impennis, the Imperial Woodpecker Campehilus imperialis, the Huia Heteralocha acutirostris, the Kauai Oo Moho braccathus and the South Island Kokako Callaeas cinereus), there were partial mitochondrial sequences available for comparison. For all seven species, we found sequence similarities of >98%. For the remaining four species (the Kamao Myadestes myadestinus, the Paradise Parrot Psephotellus pulcherrimus, the Ou Psittirostra psittacea and the Lesser Akialoa Akialoa obscura), there was no sequence information available for comparison, so we conducted blast searches and phylogenetic analyses to determine their phylogenetic positions and identify their closest extant relatives. These mitogenomes will be valuable for future analyses of avian phylogenetics and illustrate the importance of museum collections as repositories for genomics resources.     

Characterizing DNA preservation in degraded specimens of Amara alpina (Carabidae: Coleoptera)

DNA preserved in degraded beetle (Coleoptera) specimens, including those derived from dry‐stored museum and ancient permafrost‐preserved environments, could provide a valuable resource for researchers interested in species and population histories over timescales from decades to millenia. However, the potential of these samples as genetic resources is currently unassessed. Here, using Sanger and Illumina shotgun sequence data, we explored DNA preservation in specimens of the ground beetle Amara alpina, from both museum and ancient environments. Nearly all museum specimens had amplifiable DNA, with the maximum amplifiable fragment length decreasing with age. Amplification of DNA was only possible in 45% of ancient specimens. Preserved mitochondrial DNA fragments were significantly longer than those of nuclear DNA in both museum and ancient specimens. Metagenomic characterization of extracted DNA demonstrated that parasite‐derived sequences, including Wolbachia and Spiroplasma, are recoverable from museum beetle specimens. Ancient DNA extracts contained beetle DNA in amounts comparable to museum specimens. Overall, our data demonstrate that there is great potential for both museum and ancient specimens of beetles in future genetic studies, and we see no reason why this would not be the case for other orders of insect.     

From genomics to functional markers in the era of next-generation sequencing

The availability of complete genome sequences, along with other genomic resources for Arabidopsis, rice, pigeon pea, soybean and other crops, has revolutionized our understanding of the genetic make-up of plants. Next-generation DNA sequencing (NGS) has facilitated single nucleotide polymorphism discovery in plants. Functionally-characterized sequences can be identified and functional markers (FMs) for important traits can be developed at an ever-increasing ease. FMs are derived from sequence polymorphisms found in allelic variants of a functional gene. Linkage disequilibrium-based association mapping and homologous recombinants have been developed for identification of “perfect” markers for their use in crop improvement practices. Compared with many other molecular markers, FMs derived from the functionally characterized sequence genes using NGS techniques and their use provide opportunities to develop high-yielding plant genotypes resistant to various stresses at a fast pace.     

Rapid hybrid de novo assembly of a microbial genome using only short reads: Corynebacterium pseudotuberculosis I19 as a case study

Due to the advent of the so-called Next-Generation Sequencing (NGS) technologies the amount of monetary and temporal resources for whole-genome sequencing has been reduced by several orders of magnitude. Sequence reads can be assembled either by anchoring them directly onto an available reference genome (classical reference assembly), or can be concatenated by overlap (de novo assembly). The latter strategy is preferable because it tends to maintain the architecture of the genome sequence the however, depending on the NGS platform used, the shortness of read lengths cause tremendous problems the in the subsequent genome assembly phase, impeding closing of the entire genome sequence. To address the problem, we developed a multi-pronged hybrid de novo strategy combining De Bruijn graph and Overlap-Layout-Consensus methods, which was used to assemble from short reads the entire genome of Corynebacterium pseudotuberculosis strain I19, a bacterium with immense importance in veterinary medicine that causes Caseous Lymphadenitis in ruminants, principally ovines and caprines. Briefly, contigs were assembled de novo from the short reads and were only oriented using a reference genome by anchoring. Remaining gaps were closed using iterative anchoring of short reads by craning to gap flanks. Finally, we compare the genome sequence assembled using our hybrid strategy to a classical reference assembly using the same data as input and show that with the availability of a reference genome, it pays off to use the hybrid de novo strategy, rather than a classical reference assembly, because more genome sequences are preserved using the former.     

Genomic markers on synthetic genomes

Genome synthesis endows scientists the ability of de novo creating genomes absent in nature, by thorough redesigning DNA sequences and introducing numerous custom features. However, the genome synthesis is a labor‐ and time‐consuming work, and thus it is a challenge to verify and quantify the synthetic genome rapidly and precisely. Thus, specific DNA sequences different from native genomic sequences are designed into synthetic genomes during synthesis, namely genomic markers. Genomic markers can be easily detected by PCR reaction, whole‐genome sequencing (WGS) and a variety of methods to identify the synthetic genome from native one. Here, we review types and applications of genomic markers utilized in synthetic genomes, with the hope of providing a guidance for future works.     

Biased distribution of DNA uptake sequences towards genome maintenance genes

Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9–10mers residing within coding regions are the DNA uptake sequences (DUS) required for natural genetic transformation. More importantly, we found a significantly higher density of DUS within genes involved in DNA repair, recombination, restriction-modification and replication than in any other annotated gene group in these organisms. Pasteurella multocida also displayed high frequencies of a putative DUS identical to that previously identified in H.influenzae and with a skewed distribution towards genome maintenance genes, indicating that this bacterium might be transformation competent under certain conditions. These results imply that the high frequency of DUS in genome maintenance genes is conserved among phylogenetically divergent species and thus are of significant biological importance. Increased DUS density is expected to enhance DNA uptake and the over-representation of DUS in genome maintenance genes might reflect facilitated recovery of genome preserving functions. For example, transient and beneficial increase in genome instability can be allowed during pathogenesis simply through loss of antimutator genes, since these DUS-containing sequences will be preferentially recovered. Furthermore, uptake of such genes could provide a mechanism for facilitated recovery from DNA damage after genotoxic stress.