A Complete Mitochondrial Genome Sequence from a Mesolithic Wild Aurochs (Bos primigenius)

Background

The derivation of domestic cattle from the extinct wild aurochs (Bos primigenius) has been well-documented by archaeological and genetic studies. Genetic studies point towards the Neolithic Near East as the centre of origin for Bos taurus, with some lines of evidence suggesting possible, albeit rare, genetic contributions from locally domesticated wild aurochsen across Eurasia. Inferences from these investigations have been based largely on the analysis of partial mitochondrial DNA sequences generated from modern animals, with limited sequence data from ancient aurochsen samples. Recent developments in DNA sequencing technologies, however, are affording new opportunities for the examination of genetic material retrieved from extinct species, providing new insight into their evolutionary history. Here we present DNA sequence analysis of the first complete mitochondrial genome (16,338 base pairs) from an archaeologically-verified and exceptionally-well preserved aurochs bone sample.

Methodology

DNA extracts were generated from an aurochs humerus bone sample recovered from a cave site located in Derbyshire, England and radiocarbon-dated to 6,738±68 calibrated years before present. These extracts were prepared for both Sanger and next generation DNA sequencing technologies (Illumina Genome Analyzer). In total, 289.9 megabases (22.48%) of the post-filtered DNA sequences generated using the Illumina Genome Analyzer from this sample mapped with confidence to the bovine genome. A consensus B. primigenius mitochondrial genome sequence was constructed and was analysed alongside all available complete bovine mitochondrial genome sequences.

Conclusions

For all nucleotide positions where both Sanger and Illumina Genome Analyzer sequencing methods gave high-confidence calls, no discrepancies were observed. Sequence analysis reveals evidence of heteroplasmy in this sample and places this mitochondrial genome sequence securely within a previously identified aurochsen haplogroup (haplogroup P), thus providing novel insights into pre-domestic patterns of variation. The high proportion of authentic, endogenous aurochs DNA preserved in this sample bodes well for future efforts to determine the complete genome sequence of a wild ancestor of domestic cattle.     

Enrichment of genomic DNA for polymorphism detection in a non-model highly polyploid crop plant

Large polyploid genomes of non-model species remain challenging targets for DNA polymorphism discovery despite the increasing throughput and continued reductions in cost of sequencing with new technologies. For these species especially, there remains a requirement to enrich genomic DNA to discover polymorphisms in regions of interest because of large genome size and to provide the sequence depth to enable estimation of copy number. Various methods of enriching DNA have been utilised, but some recent methods enable the efficient sampling of large regions (e.g. the exome). We have utilised one of these methods, solution-based hybridization (Agilent SureSelect), to capture regions of the genome of two sugarcane genotypes (one Saccharum officinarum and one Saccharum hybrid) based mainly on gene sequences from the close relative Sorghum bicolor. The capture probes span approximately 5.8?megabases (Mb). The enrichment over whole-genome shotgun sequencing was 10-11-fold for the two genotypes tested. This level of enrichment has important consequences for detecting single nucleotide polymorphisms (SNPs) from a single lane of Illumina (Genome Analyzer) sequence reads. The detection of polymorphisms was enabled by the depth of sequence at or near probe sites and enabled the detection of 270?000-280?000 SNPs within each genotype from a single lane of sequence using stringent detection parameters. The SNPs were present in 13?000-16?000 targeted genes, which would enable mapping of a large number of these chosen genes. SNP validation from 454 sequencing and between-genotype confirmations gave an 87%-91% validation rate.     

High-performance single-chip exon capture allows accurate whole exome sequencing using the Illumina Genome Analyzer

Here we present an adaptation of NimbleGen 2.1M-probe array sequence capture for whole exome sequencing using the Illumina Genome Analyzer (GA) platform. The protocol involves two-stage library construction. The specificity of exome enrichment was approximately 80% with 95.6% even coverage of the 34 Mb target region at an average sequencing depth of 33-fold. Comparison of our results with whole genome shot-gun resequencing results showed that the exome SNP calls gave only 0.97% false positive and 6.27% false negative variants. Our protocol is also well suited for use with whole genome amplified DNA. The results presented here indicate that there is a promising future for large-scale population genomics and medical studies using a whole exome sequencing approach.     

Hybrid selection for sequencing pathogen genomes from clinical samples

We have adapted a solution hybrid selection protocol to enrich pathogen DNA in clinical samples dominated by human genetic material. Using mock mixtures of human and Plasmodium falciparum malaria parasite DNA as well as clinical samples from infected patients, we demonstrate an average of approximately 40-fold enrichment of parasite DNA after hybrid selection. This approach will enable efficient genome sequencing of pathogens from clinical samples, as well as sequencing of endosymbiotic organisms such as Wolbachia that live inside diverse metazoan phyla.     

A Method Enabling High-Throughput Sequencing of Human Cytomegalovirus Complete Genomes from Clinical Isolates

Human cytomegalovirus (HCMV) is a ubiquitous virus that can cause serious sequelae in immunocompromised patients and in the developing fetus. The coding capacity of the 235 kbp genome is still incompletely understood, and there is a pressing need to characterize genomic contents in clinical isolates. In this study, a procedure for the high-throughput generation of full genome consensus sequences from clinical HCMV isolates is presented. This method relies on low number passaging of clinical isolates on human fibroblasts, followed by digestion of cellular DNA and purification of viral DNA. After multiple displacement amplification, highly pure viral DNA is generated. These extracts are suitable for high-throughput next-generation sequencing and assembly of consensus sequences. Throughout a series of validation experiments, we showed that the workflow reproducibly generated consensus sequences representative for the virus population present in the original clinical material. Additionally, the performance of 454 GS FLX and/or Illumina Genome Analyzer datasets in consensus sequence deduction was evaluated. Based on assembly performance data, the Illumina Genome Analyzer was the platform of choice in the presented workflow. Analysis of the consensus sequences derived in this study confirmed the presence of gene-disrupting mutations in clinical HCMV isolates independent from in vitro passaging. These mutations were identified in genes RL5A, UL1, UL9, UL111A and UL150. In conclusion, the presented workflow provides opportunities for high-throughput characterization of complete HCMV genomes that could deliver new insights into HCMV coding capacity and genetic determinants of viral tropism and pathogenicity.     

Variant Identification in Multi-Sample Pools by Illumina Genome Analyzer Sequencing

Multi-sample pooling and Illumina Genome Analyzer (GA) sequencing allows high throughput sequencing of multiple samples to determine population sequence variation. A preliminary experiment, using the RET proto-oncogene as a model, predicted ≤30 samples could be pooled to reliably detect singleton variants without requiring additional confirmation testing. This report used 30 and 50 sample pools to test the hypothesized pooling limit and also to test recent protocol improvements, Illumina GAIIx upgrades, and longer read chemistry. The SequalPrep^TM method was used to normalize amplicons before pooling. For comparison, a single ‘control’ sample was run in a different flow cell lane. Data was evaluated by variant read percentages and the subtractive correction method which utilizes the control sample. In total, 59 variants were detected within the pooled samples, which included all 47 known true variants. The 15 known singleton variants due to Sanger sequencing had an average of 1.62±0.26% variant reads for the 30 pool (expected 1.67% for a singleton variant [unique variant within the pool]) and 1.01±0.19% for the 50 pool (expected 1%). The 76 base read lengths had higher error rates than shorter read lengths (33 and 50 base reads), which eliminated the distinction of true singleton variants from background error. This report demonstrated pooling limits from 30 up to 50 samples (depending on error rates and coverage), for reliable singleton variant detection. The presented pooling protocols and analysis methods can be used for variant discovery in other genes, facilitating molecular diagnostic test design and interpretation.     

Performance of microarray and liquid based capture methods for target enrichment for massively parallel sequencing and SNP discovery

Targeted sequencing is a cost-efficient way to obtain answers to biological questions in many projects, but the choice of the enrichment method to use can be difficult. In this study we compared two hybridization methods for target enrichment for massively parallel sequencing and single nucleotide polymorphism (SNP) discovery, namely Nimblegen sequence capture arrays and the SureSelect liquid-based hybrid capture system. We prepared sequencing libraries from three HapMap samples using both methods, sequenced the libraries on the Illumina Genome Analyzer, mapped the sequencing reads back to the genome, and called variants in the sequences. 74-75% of the sequence reads originated from the targeted region in the SureSelect libraries and 41-67% in the Nimblegen libraries. We could sequence up to 99.9% and 99.5% of the regions targeted by capture probes from the SureSelect libraries and from the Nimblegen libraries, respectively. The Nimblegen probes covered 0.6 Mb more of the original 3.1 Mb target region than the SureSelect probes. In each sample, we called more SNPs and detected more novel SNPs from the libraries that were prepared using the Nimblegen method. Thus the Nimblegen method gave better results when judged by the number of SNPs called, but this came at the cost of more over-sampling.     

Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology

Organellar DNA sequences are widely used in evolutionary and population genetic studies, however, the conservative nature of chloroplast gene and genome evolution often limits phylogenetic resolution and statistical power. To gain maximal access to the historical record contained within chloroplast genomes, we have adapted multiplex sequencing-by-synthesis (MSBS) to simultaneously sequence multiple genomes using the Illumina Genome Analyzer. We PCR-amplified ~120 kb plastomes from eight species (seven Pinus, one Picea) in 35 reactions. Pooled products were ligated to modified adapters that included 3 bp indexing tags and samples were multiplexed at four genomes per lane. Tagged microreads were assembled by de novo and reference-guided assembly methods, using previously published Pinus plastomes as surrogate references. Assemblies for these eight genomes are estimated at 88–94% complete, with an average sequence depth of 55× to 186×. Mononucleotide repeats interrupt contig assembly with increasing repeat length, and we estimate that the limit for their assembly is 16 bp. Comparisons to 37 kb of Sanger sequence show a validated error rate of 0.056%, and conspicuous errors are evident from the assembly process. This efficient sequencing approach yields high-quality draft genomes and should have immediate applicability to genomes with comparable complexity.     

Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample

Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ~90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R(2)>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ~3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.     

Identification of genetic variants using bar-coded multiplexed sequencing

We developed a generalized framework for multiplexed resequencing of targeted human genome regions on the Illumina Genome Analyzer using degenerate indexed DNA bar codes ligated to fragmented DNA before sequencing. Using this method, we simultaneously sequenced the DNA of multiple HapMap individuals at several Encyclopedia of DNA Elements (ENCODE) regions. We then evaluated the use of Bayes factors for discovering and genotyping polymorphisms. For polymorphisms that were either previously identified within the Single Nucleotide Polymorphism database (dbSNP) or visually evident upon re-inspection of archived ENCODE traces, we observed a false positive rate of 11.3% using strict thresholds for predicting variants and 69.6% for lax thresholds. Conversely, false negative rates were 10.8-90.8%, with false negatives at stricter cut-offs occurring at lower coverage (<10 aligned reads). These results suggest that >90% of genetic variants are discoverable using multiplexed sequencing provided sufficient coverage at the polymorphic base.     

Genome data: what do we learn?

Genome sequence information has continued to accumulate at a spectacular pace during the past year. Details of the sequence and gene content of human chromosome 22 were published. The sequencing and annotation of the first two Arabidopsis thaliana chromosomes was completed. The sequence of chromosome 3 from Plasmodium falciparum, the second sequenced malaria chromosome, was reported, as was that of chromosome 1 from Leishmania major. The complete genomic sequences of five microbes were reported. Approaches to using data from completely sequenced microbial genomes in phylogenetic studies are being explored, as is the application of microarrays to whole genome expression analysis.     

Comparison of sequence reads obtained from three next-generation sequencing platforms

Next-generation sequencing technologies enable the rapid cost-effective production of sequence data. To evaluate the performance of these sequencing technologies, investigation of the quality of sequence reads obtained from these methods is important. In this study, we analyzed the quality of sequence reads and SNP detection performance using three commercially available next-generation sequencers, i.e., Roche Genome Sequencer FLX System (FLX), Illumina Genome Analyzer (GA), and Applied Biosystems SOLiD system (SOLiD). A common genomic DNA sample obtained from Escherichia coli strain DH1 was applied to these sequencers. The obtained sequence reads were aligned to the complete genome sequence of E. coli DH1, to evaluate the accuracy and sequence bias of these sequence methods. We found that the fraction of "junk" data, which could not be aligned to the reference genome, was largest in the data set of SOLiD, in which about half of reads could not be aligned. Among data sets after alignment to the reference, sequence accuracy was poorest in GA data sets, suggesting relatively low fidelity of the elongation reaction in the GA method. Furthermore, by aligning the sequence reads to the E. coli strain W3110, we screened sequence differences between two E. coli strains using data sets of three different next-generation platforms. The results revealed that the detected sequence differences were similar among these three methods, while the sequence coverage required for the detection was significantly small in the FLX data set. These results provided valuable information on the quality of short sequence reads and the performance of SNP detection in three next-generation sequencing platforms.     

Comparison of the Illumina Genome Analyzer and Roche 454 GS FLX for Resequencing of Hypertrophic Cardiomyopathy-Associated Genes

Next-generation sequencing (NGS) is widely used in biomedical research, but its adoption has been limited in molecular diagnostics. One application of NGS is the targeted resequencing of genes whose mutations lead to an overlapping clinical phenotype. This study evaluated the comparative performance of the Illumina Genome Analyzer and Roche 454 GS FLX for the resequencing of 16 genes associated with hypertrophic cardiomyopathy (HCM). Using a single human genomic DNA sample enriched by long-range PCR (LR-PCR), 40 GS FLX and 31 Genome Analyzer exon variants were identified using ≥30-fold read-coverage and ≥20% read-percentage selection criteria. Twenty-seven platform concordant variants were Sanger-confirmed. The discordant variants segregated into two categories: variants with read coverages ≥30 on one platform but <30-fold on the alternate platform and variants with read percentages ≥20% on one platform but <20% on the alternate platform. All variants with <30-fold coverage were Sanger-confirmed, suggesting that the coverage criterion of ≥30-fold is too stringent for variant discovery. The variants with <20% read percentage were identified as reference sequence based on Sanger sequencing. These variants were found in homopolymer tracts and short-read misalignments, specifically in genes with high identity. The results of the current study demonstrate the feasibility of combining LR-PCR with the Genome Analyzer or GS FLX for targeted resequencing of HCM-associated genes.     

Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling

Genome-wide mapping of 5-methylcytosine is of broad interest to many fields of biology and medicine. A variety of methods have been developed, and several have recently been advanced to genome-wide scale using arrays and next-generation sequencing approaches. We have previously reported reduced representation bisulfite sequencing (RRBS), a bisulfite-based protocol that enriches CG-rich parts of the genome, thereby reducing the amount of sequencing required while capturing the majority of promoters and other relevant genomic regions. The approach provides single-nucleotide resolution, is highly sensitive and provides quantitative DNA methylation measurements. This protocol should enable any standard molecular biology laboratory to generate RRBS libraries of high quality. Briefly, purified genomic DNA is digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. After end-repair, A-tailing and ligation to methylated Illumina adapters, the CpG-rich DNA fragments (40-220 bp) are size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome Analyzer. Note that alignment and analysis of RRBS sequencing reads are not covered in this protocol. The extremely low input requirements (10-300 ng), the applicability of the protocol to formalin-fixed and paraffin-embedded samples, and the technique's single-nucleotide resolution extends RRBS to a wide range of biological and clinical samples and research applications. The entire process of RRBS library construction takes ～9 d.     

Plasmodium knowlesi Genome Sequences from Clinical Isolates Reveal Extensive Genomic Dimorphism

Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology.     

SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries

High-density single-nucleotide polymorphism (SNP) arrays have revolutionized the ability of genome-wide association studies to detect genomic regions harboring sequence variants that affect complex traits. Extensive numbers of validated SNPs with known allele frequencies are essential to construct genotyping assays with broad utility. We describe an economical, efficient, single-step method for SNP discovery, validation and characterization that uses deep sequencing of reduced representation libraries (RRLs) from specified target populations. Using nearly 50 million sequences generated on an Illumina Genome Analyzer from DNA of 66 cattle representing three populations, we identified 62,042 putative SNPs and predicted their allele frequencies. Genotype data for these 66 individuals validated 92% of 23,357 selected genome-wide SNPs, with a genotypic and sequence allele frequency correlation of r = 0.67. This approach for simultaneous de novo discovery of high-quality SNPs and population characterization of allele frequencies may be applied to any species with at least a partially sequenced genome.     

Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven’t benefited much from those next-generation sequencing technolo...     

Alta-Cyclic: a self-optimizing base caller for next-generation sequencing

Next-generation sequencing is limited to short read lengths and by high error rates. We systematically analyzed sources of noise in the Illumina Genome Analyzer that contribute to these high error rates and developed a base caller, Alta-Cyclic, that uses machine learning to compensate for noise factors. Alta-Cyclic substantially improved the number of accurate reads for sequencing runs up to 78 bases and reduced systematic biases, facilitating confident identification of sequence variants.     

Automated sequencing of complete mitochondrial genomes from laser-capture microdissected samples

Mitochondrial DNA mutations have been related to both aging and a variety of diseases such as cancer. Due to the relatively small size of the genome (16 kb) and with the use of automated DNA sequencing, the entire genome can be sequenced from clinical specimens in days. We present a reliable approach to complete mitochondrial genome sequencing from laser-capture microdissected human clinical cancer specimens that overcome the inherent limitations of relatively small tissue samples and partial DNA degradation, which are unavoidable when laser-capture microdissection is used to attain pure populations of cells from heterogeneous tissues obtained from surgical procedures. The acquisition of sufficient template combined with a standard set of 18 pairs of PCR primers allows for the efficient amplification of the genome. Subsequent single-stranded amplification is performed using 36 sequencing primers, and samples are run on an ABI PRISM 3100 Genetic Analyzer. The use of this procedure should allow even investigators with little experience sequencing from clinical specimens success in complete mitochondrial genome sequencing.     

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.