Probabilistic base calling of Solexa sequencing data

Background  

Solexa/Illumina short-read ultra-high throughput DNA sequencing technology produces millions of short tags (up to 36 bases) by parallel sequencing-by-synthesis of DNA colonies. The processing and statistical analysis of such high-throughput data poses new challenges; currently a fair proportion of the tags are routinely discarded due to an inability to match them to a reference sequence, thereby reducing the effective throughput of the technology.     

SASI-Seq: sample assurance Spike-Ins,and highly differentiating 384 barcoding for Illumina sequencing

Background

A minor but significant fraction of samples subjected to next-generation sequencing methods are either mixed-up or cross-contaminated. These events can lead to false or inconclusive results. We have therefore developed SASI-Seq; a process whereby a set of uniquely barcoded DNA fragments are added to samples destined for sequencing. From the final sequencing data, one can verify that all the reads derive from the original sample(s) and not from contaminants or other samples.

Results

By adding a mixture of three uniquely barcoded amplicons, of different sizes spanning the range of insert sizes one would normally use for Illumina sequencing, at a spike-in level of approximately 0.1%, we demonstrate that these fragments remain intimately associated with the sample. They can be detected following even the tightest size selection regimes or exome enrichment and can report the occurrence of sample mix-ups and cross-contamination.As a consequence of this work, we have designed a set of 384 eleven-base Illumina barcode sequences that are at least 5 changes apart from each other, allowing for single-error correction and very low levels of barcode misallocation due to sequencing error.

Conclusion

SASI-Seq is a simple, inexpensive and flexible tool that enables sample assurance, allows deconvolution of sample mix-ups and reports levels of cross-contamination between samples throughout NGS workflows.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-110) contains supplementary material, which is available to authorized users.     

An improved chloroplast DNA extraction procedure for whole plastid genome sequencing

Background

Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.

Methodology/Principal Findings

We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method.

Conclusion

Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.     

Characterizing and measuring bias in sequence data

Background

DNA sequencing technologies deviate from the ideal uniform distribution of reads. These biases impair scientific and medical applications. Accordingly, we have developed computational methods for discovering, describing and measuring bias.

Results

We applied these methods to the Illumina, Ion Torrent, Pacific Biosciences and Complete Genomics sequencing platforms, using data from human and from a set of microbes with diverse base compositions. As in previous work, library construction conditions significantly influence sequencing bias. Pacific Biosciences coverage levels are the least biased, followed by Illumina, although all technologies exhibit error-rate biases in high- and low-GC regions and at long homopolymer runs. The GC-rich regions prone to low coverage include a number of human promoters, so we therefore catalog 1,000 that were exceptionally resistant to sequencing. Our results indicate that combining data from two technologies can reduce coverage bias if the biases in the component technologies are complementary and of similar magnitude. Analysis of Illumina data representing 120-fold coverage of a well-studied human sample reveals that 0.20% of the autosomal genome was covered at less than 10% of the genome-wide average. Excluding locations that were similar to known bias motifs or likely due to sample-reference variations left only 0.045% of the autosomal genome with unexplained poor coverage.

Conclusions

The assays presented in this paper provide a comprehensive view of sequencing bias, which can be used to drive laboratory improvements and to monitor production processes. Development guided by these assays should result in improved genome assemblies and better coverage of biologically important loci.     

Interpretation of custom designed Illumina genotype cluster plots for targeted association studies and next-generation sequence validation

Background

High-throughput custom designed genotyping arrays are a valuable resource for biologically focused research studies and increasingly for validation of variation predicted by next-generation sequencing (NGS) technologies. We investigate the Illumina GoldenGate chemistry using custom designed VeraCode and sentrix array matrix (SAM) assays for each of these applications, respectively. We highlight applications for interpretation of Illumina generated genotype cluster plots to maximise data inclusion and reduce genotyping errors.

Findings

We illustrate the dramatic effect of outliers in genotype calling and data interpretation, as well as suggest simple means to avoid genotyping errors. Furthermore we present this platform as a successful method for two-cluster rare or non-autosomal variant calling. The success of high-throughput technologies to accurately call rare variants will become an essential feature for future association studies. Finally, we highlight additional advantages of the Illumina GoldenGate chemistry in generating unusually segregated cluster plots that identify potential NGS generated sequencing error resulting from minimal coverage.

Conclusions

We demonstrate the importance of visually inspecting genotype cluster plots generated by the Illumina software and issue warnings regarding commonly accepted quality control parameters. In addition to suggesting applications to minimise data exclusion, we propose that the Illumina cluster plots may be helpful in identifying potential in-put sequence errors, particularly important for studies to validate NGS generated variation.

     

WebCARMA: a web application for the functional and taxonomic classification of unassembled metagenomic reads

Background  

Metagenomics is a new field of research on natural microbial communities. High-throughput sequencing techniques like 454 or Solexa-Illumina promise new possibilities as they are able to produce huge amounts of data in much shorter time and with less efforts and costs than the traditional Sanger technique. But the data produced comes in even shorter reads (35-100 basepairs with Illumina, 100-500 basepairs with 454-sequencing). CARMA is a new software pipeline for the characterisation of species composition and the genetic potential of microbial samples using short, unassembled reads.     

Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

Background  

Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship.     

iMSAT: a novel approach to the development of microsatellite loci using barcoded Illumina libraries

Background

Illumina sequencing with its high number of reads and low per base pair cost is an attractive technology for development of molecular resources for non-model organisms. While many software packages have been developed to identify short tandem repeats (STRs) from next-generation sequencing data, these methods do not inform the investigator as to whether or not candidate loci are polymorphic in their target populations.

Results

We provide a python program iMSAT that uses the polymorphism data obtained from mapping individual Illumina sequence reads onto a reference genome to identify polymorphic STRs. Using this approach, we identified 9,119 candidate polymorphic STRs for use with the parasitoid wasp Trioxys pallidus and 2,378 candidate polymorphic STRs for use with the aphid Chromaphis juglandicola. For both organisms we selected 20 candidate tri-nucleotide STRs for validation. Using fluorescent-labeled oligonucleotide primers, we genotyped 91 female T. pallidus collected in nine localities and 46 female C. juglandicola collected in 4 localities and found 15 of the examined markers to be polymorphic for T. pallidus and 12 of the examined markers to be polymorphic for C. juglandicola.

Conclusions

We present a novel approach that uses standard Illumina barcoding primers and a single Illumina HiSeq run to target polymorphic STR fragments to develop and test STR markers. We validate this approach using the parasitoid wasp T. pallidus and its aphid host C. juglandicola. This approach, which would also be compatible with 454 Sequencing, allowed us to quickly identify markers with known variability. Accordingly, our method constitutes a significant improvement over existing STR identification software packages.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-858) contains supplementary material, which is available to authorized users.     

Validation of multiple single nucleotide variation calls by additional exome analysis with a semiconductor sequencer to supplement data of whole-genome sequencing of a human population

Background

Validation of single nucleotide variations in whole-genome sequencing is critical for studying disease-related variations in large populations. A combination of different types of next-generation sequencers for analyzing individual genomes may be an efficient means of validating multiple single nucleotide variations calls simultaneously.

Results

Here, we analyzed 12 independent Japanese genomes using two next-generation sequencing platforms: the Illumina HiSeq 2500 platform for whole-genome sequencing (average depth 32.4×), and the Ion Proton semiconductor sequencer for whole exome sequencing (average depth 109×). Single nucleotide polymorphism (SNP) calls based on the Illumina Human Omni 2.5-8 SNP chip data were used as the reference. We compared the variant calls for the 12 samples, and found that the concordance between the two next-generation sequencing platforms varied between 83% and 97%.

Conclusions

Our results show the versatility and usefulness of the combination of exome sequencing with whole-genome sequencing in studies of human population genetics and demonstrate that combining data from multiple sequencing platforms is an efficient approach to validate and supplement SNP calls.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-673) contains supplementary material, which is available to authorized users.     

The Fast Changing Landscape of Sequencing Technologies and Their Impact on Microbial Genome Assemblies and Annotation

Background

The emergence of next generation sequencing (NGS) has provided the means for rapid and high throughput sequencing and data generation at low cost, while concomitantly creating a new set of challenges. The number of available assembled microbial genomes continues to grow rapidly and their quality reflects the quality of the sequencing technology used, but also of the analysis software employed for assembly and annotation.

Methodology/Principal Findings

In this work, we have explored the quality of the microbial draft genomes across various sequencing technologies. We have compared the draft and finished assemblies of 133 microbial genomes sequenced at the Department of Energy-Joint Genome Institute and finished at the Los Alamos National Laboratory using a variety of combinations of sequencing technologies, reflecting the transition of the institute from Sanger-based sequencing platforms to NGS platforms. The quality of the public assemblies and of the associated gene annotations was evaluated using various metrics. Results obtained with the different sequencing technologies, as well as their effects on downstream processes, were analyzed. Our results demonstrate that the Illumina HiSeq 2000 sequencing system, the primary sequencing technology currently used for de novo genome sequencing and assembly at JGI, has various advantages in terms of total sequence throughput and cost, but it also introduces challenges for the downstream analyses. In all cases assembly results although on average are of high quality, need to be viewed critically and consider sources of errors in them prior to analysis.

Conclusion

These data follow the evolution of microbial sequencing and downstream processing at the JGI from draft genome sequences with large gaps corresponding to missing genes of significant biological role to assemblies with multiple small gaps (Illumina) and finally to assemblies that generate almost complete genomes (Illumina+PacBio).     

Using quality scores and longer reads improves accuracy of Solexa read mapping

Background  

Second-generation sequencing has the potential to revolutionize genomics and impact all areas of biomedical science. New technologies will make re-sequencing widely available for such applications as identifying genome variations or interrogating the oligonucleotide content of a large sample (e.g. ChIP-sequencing). The increase in speed, sensitivity and availability of sequencing technology brings demand for advances in computational technology to perform associated analysis tasks. The Solexa/Illumina 1G sequencer can produce tens of millions of reads, ranging in length from ~25–50 nt, in a single experiment. Accurately mapping the reads back to a reference genome is a critical task in almost all applications. Two sources of information that are often ignored when mapping reads from the Solexa technology are the 3' ends of longer reads, which contain a much higher frequency of sequencing errors, and the base-call quality scores.     

The cervical microbiome over 7 years and a comparison of methodologies for its characterization

Background

The rapidly expanding field of microbiome studies offers investigators a large choice of methods for each step in the process of determining the microorganisms in a sample. The human cervicovaginal microbiome affects female reproductive health, susceptibility to and natural history of many sexually transmitted infections, including human papillomavirus (HPV). At present, long-term behavior of the cervical microbiome in early sexual life is poorly understood.

Methods

The V6 and V6–V9 regions of the 16S ribosomal RNA gene were amplified from DNA isolated from exfoliated cervical cells. Specimens from 10 women participating in the Natural History Study of HPV in Guanacaste, Costa Rica were sampled successively over a period of 5–7 years. We sequenced amplicons using 3 different platforms (Sanger, Roche 454, and Illumina HiSeq 2000) and analyzed sequences using pipelines based on 3 different classification algorithms (usearch, RDP Classifier, and pplacer).

Results

Usearch and pplacer provided consistent microbiome classifications for all sequencing methods, whereas RDP Classifier deviated significantly when characterizing Illumina reads. Comparing across sequencing platforms indicated 7%–41% of the reads were reclassified, while comparing across software pipelines reclassified up to 32% of the reads. Variability in classification was shown not to be due to a difference in read lengths. Six cervical microbiome community types were observed and are characterized by a predominance of either G. vaginalis or Lactobacillus spp. Over the 5–7 year period, subjects displayed fluctuation between community types. A PERMANOVA analysis on pairwise Kantorovich-Rubinstein distances between the microbiota of all samples yielded an F-test ratio of 2.86 (p<0.01), indicating a significant difference comparing within and between subjects’ microbiota.

Conclusions

Amplification and sequencing methods affected the characterization of the microbiome more than classification algorithms. Pplacer and usearch performed consistently with all sequencing methods. The analyses identified 6 community types consistent with those previously reported. The long-term behavior of the cervical microbiome indicated that fluctuations were subject dependent.     

Development of a low bias method for characterizing viral populations using next generation sequencing technology

Background

With an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies.

Methodology/Principal Findings

We report a novel experimental method for amplifying full-length HIV genomes without the use of sequence-specific primers for high throughput DNA sequencing, followed by assembly of full length viral genome sequences from the resulting large dataset. Illumina was chosen for sequencing due to its ability to provide greater coverage of the HIV genome compared to prior methods, allowing for more comprehensive characterization of the heterogeneity present in the HIV samples analyzed. Our novel amplification method in combination with Illumina sequencing was used to analyze two HIV populations: a homogenous HIV population based on the canonical NL4-3 strain and a heterogeneous viral population obtained from a HIV patient''s infected T cells. In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity.

Significance

This study demonstrates how a lower bias amplification method in combination with next generation DNA sequencing provides in-depth, complete coverage of the HIV genome, enabling a stronger characterization of the quasispecies present in a clinically relevant HIV population as well as future study of how HIV mutates in response to a selective pressure.     

Normalization of Illumina Infinium whole-genome SNP data improves copy number estimates and allelic intensity ratios

Background  

Illumina Infinium whole genome genotyping (WGG) arrays are increasingly being applied in cancer genomics to study gene copy number alterations and allele-specific aberrations such as loss-of-heterozygosity (LOH). Methods developed for normalization of WGG arrays have mostly focused on diploid, normal samples. However, for cancer samples genomic aberrations may confound normalization and data interpretation. Therefore, we examined the effects of the conventionally used normalization method for Illumina Infinium arrays when applied to cancer samples.     

Genomic profiling of rectal adenoma and carcinoma by array-based comparative genomic hybridization

Background

Aortopathies are a group of disorders characterized by aneurysms, dilation, and tortuosity of the aorta. Because of the phenotypic overlap and genetic heterogeneity of diseases featuring aortopathy, molecular testing is often required for timely and correct diagnosis of affected individuals. In this setting next generation sequencing (NGS) offers several advantages over traditional molecular techniques.

Methods

The purpose of our study was to compare NGS enrichment methods for a clinical assay targeting the nine genes known to be associated with aortopathy. RainDance emulsion PCR and SureSelect RNA-bait hybridization capture enrichment methods were directly compared by enriching DNA from eight samples. Enriched samples were barcoded, pooled, and sequenced on the Illumina HiSeq2000 platform. Depth of coverage, consistency of coverage across samples, and the overlap of variants identified were assessed. This data was also compared to whole-exome sequencing data from ten individuals.

Results

Read depth was greater and less variable among samples that had been enriched using the RNA-bait hybridization capture enrichment method. In addition, samples enriched by hybridization capture had fewer exons with mean coverage less than 10, reducing the need for followup Sanger sequencing. Variants sets produced were 77% concordant, with both techniques yielding similar numbers of discordant variants.

Conclusions

When comparing the design flexibility, performance, and cost of the targeted enrichment methods to whole-exome sequencing, the RNA-bait hybridization capture enrichment gene panel offers the better solution for interrogating the aortopathy genes in a clinical laboratory setting.     

Rapid sequencing of the bamboo mitochondrial genome using Illumina technology and parallel episodic evolution of organelle genomes in grasses

Background

Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change.

Methodology/Principal Findings

We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the well-resolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significant rate change (∼4-fold) in mt genome before and after the diversification of Poaceae both in synonymous and nonsynonymous terms. Furthermore, the rate change was correlated with that of chloroplast genomes in grasses.

Conclusions/Significance

Our result demonstrates that it is a rapid and efficient approach to obtain angiosperm mt genome sequences using Illumina sequencing technology. The parallel episodic evolution of mt and chloroplast genomes in grasses is consistent with lineage effects.     

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.     

Scalable transcriptome preparation for massive parallel sequencing

Background

The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity.

Methodology

In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation.

Conclusion/Significance

The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods.