Pseudo-Sanger sequencing: massively parallel production of long and near error-free reads using NGS technology

Background

Usually, next generation sequencing (NGS) technology has the property of ultra-high throughput but the read length is remarkably short compared to conventional Sanger sequencing. Paired-end NGS could computationally extend the read length but with a lot of practical inconvenience because of the inherent gaps. Now that Illumina paired-end sequencing has the ability of read both ends from 600 bp or even 800 bp DNA fragments, how to fill in the gaps between paired ends to produce accurate long reads is intriguing but challenging.

Results

We have developed a new technology, referred to as pseudo-Sanger (PS) sequencing. It tries to fill in the gaps between paired ends and could generate near error-free sequences equivalent to the conventional Sanger reads in length but with the high throughput of the Next Generation Sequencing. The major novelty of PS method lies on that the gap filling is based on local assembly of paired-end reads which have overlaps with at either end. Thus, we are able to fill in the gaps in repetitive genomic region correctly. The PS sequencing starts with short reads from NGS platforms, using a series of paired-end libraries of stepwise decreasing insert sizes. A computational method is introduced to transform these special paired-end reads into long and near error-free PS sequences, which correspond in length to those with the largest insert sizes. The PS construction has 3 advantages over untransformed reads: gap filling, error correction and heterozygote tolerance. Among the many applications of the PS construction is de novo genome assembly, which we tested in this study. Assembly of PS reads from a non-isogenic strain of Drosophila melanogaster yields an N50 contig of 190 kb, a 5 fold improvement over the existing de novo assembly methods and a 3 fold advantage over the assembly of long reads from 454 sequencing.

Conclusions

Our method generated near error-free long reads from NGS paired-end sequencing. We demonstrated that de novo assembly could benefit a lot from these Sanger-like reads. Besides, the characteristic of the long reads could be applied to such applications as structural variations detection and metagenomics.

Electronic supplementary material

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The Streptomyces leeuwenhoekii genome: de novo sequencing and assembly in single contigs of the chromosome,circular plasmid pSLE1 and linear plasmid pSLE2

Background

Next Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis. This approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory. Streptomyces leeuwenhoekii (formerly Streptomyces sp. strain C34) was isolated from the hyper-arid high-altitude Atacama Desert in Chile and shown to produce novel polyketide antibiotics.

Results

Here we present the de novo sequencing of the S. leeuwenhoekii linear chromosome (8 Mb) and two extrachromosomal replicons, the circular pSLE1 (86 kb) and the linear pSLE2 (132 kb), all in single contigs, obtained by combining Pacific Biosciences SMRT (PacBio) and Illumina MiSeq technologies. We identified the biosynthetic gene clusters for chaxamycin, chaxalactin, hygromycin A and desferrioxamine E, metabolites all previously shown to be produced by this strain (J Nat Prod, 2011, 74:1965) and an additional 31 putative gene clusters for specialised metabolites. As well as gene clusters for polyketides and non-ribosomal peptides, we also identified three gene clusters encoding novel lasso-peptides.

Conclusions

The S. leeuwenhoekii genome contains 35 gene clusters apparently encoding the biosynthesis of specialised metabolites, most of them completely novel and uncharacterised. This project has served to evaluate the current state of NGS for efficient and effective genome mining of high GC actinomycetes. The PacBio technology now permits the assembly of actinomycete replicons into single contigs with >99 % accuracy. The assembled Illumina sequence permitted not only the correction of omissions found in GC homopolymers in the PacBio assembly (exacerbated by the high GC content of actinomycete DNA) but it also allowed us to obtain the sequences of the termini of the chromosome and of a linear plasmid that were not assembled by PacBio. We propose an experimental pipeline that uses the Illumina assembled contigs, in addition to just the reads, to complement the current limitations of the PacBio sequencing technology and assembly software.

Electronic supplementary material

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Is the whole greater than the sum of its parts? De novo assembly strategies for bacterial genomes based on paired-end sequencing

Background

Whole genome sequence construction is becoming increasingly feasible because of advances in next generation sequencing (NGS), including increasing throughput and read length. By simply overlapping paired-end reads, we can obtain longer reads with higher accuracy, which can facilitate the assembly process. However, the influences of different library sizes and assembly methods on paired-end sequencing-based de novo assembly remain poorly understood.

Results

We used 250 bp Illumina Miseq paired-end reads of different library sizes generated from genomic DNA from Escherichia coli DH1 and Streptococcus parasanguinis FW213 to compare the assembly results of different library sizes and assembly approaches. Our data indicate that overlapping paired-end reads can increase read accuracy but sometimes cause insertion or deletions. Regarding genome assembly, merged reads only outcompete original paired-end reads when coverage depth is low, and larger libraries tend to yield better assembly results. These results imply that distance information is the most critical factor during assembly. Our results also indicate that when depth is sufficiently high, assembly from subsets can sometimes produce better results.

Conclusions

In summary, this study provides systematic evaluations of de novo assembly from paired end sequencing data. Among the assembly strategies, we find that overlapping paired-end reads is not always beneficial for bacteria genome assembly and should be avoided or used with caution especially for genomes containing high fraction of repetitive sequences. Because increasing numbers of projects aim at bacteria genome sequencing, our study provides valuable suggestions for the field of genomic sequence construction.

Electronic supplementary material

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Genome assembly using Nanopore-guided long and error-free DNA reads

Background

Long-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments.

Results

The assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting close to 30% of errors. Here, we presented a hybrid approach developed to take advantage of data generated using MinION® device. We sequenced a well-known bacterium, Acinetobacter baylyi ADP1 and applied our method to obtain a highly contiguous (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads.

Conclusions

We described NaS tool, a hybrid approach allowing the sequencing of microbial genomes using the MinION® device. Our method, based ideally on 20x and 50x of NaS and Illumina reads respectively, provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, currently with a high error rate, it is already useful in the generation of high-quality genome assemblies.

Electronic supplementary material

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Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome

Background

One of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility.

Results

Target enrichment was carried out using droplet-base multiplex polymerase chain reaction (PCR) technology (RainDance®) designed to yield amplicons averaging 1 kb fragment size from candidate 44 loci (99.8% unique base-pair coverage). The total targeted sequence was 3.18 Mb per sample. SMS was carried out using single molecule, real-time DNA sequencing (SMRT® Pacific Biosciences®), average raw read length = 1178 nucleotides, 5% of the amplicons >6000 nucleotides). After filtering with circular consensus (CCS) reads, the mean read length was 3200 nucleotides (97% CCS accuracy). Primary data analyses, alignment and filtering utilized the Pacific Biosciences® SMRT portal. Secondary analysis was conducted using the Genome Analysis Toolkit for SNP discovery l and wANNOVAR for functional analysis of variants. Filtered functional variants 18 of 19 (94.7%) were further confirmed using conventional Sanger sequencing. CCS reads were able to accurately detect zygosity. Coverage within GC rich regions (i.e.VEGFR; 72% GC rich) was achieved by capturing long genomic DNA (gDNA) fragments and reading into regions that flank the capture regions. As proof of concept, a non-synonymous LHCGR variant captured in two severe OHSS cases, and verified by conventional sequencing.

Conclusions

Combining emulsion PCR-generated 1 kb amplicons and SMRT DNA sequencing permitted greater depth of coverage for T-SMS and facilitated easier sequence assembly. To the best of our knowledge, this is the first report combining emulsion PCR and T-SMS for long reads using human DNA samples, and NGS panel designed for biomarker discovery in OHSS.

Electronic supplementary material

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SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome

Background

Third generation sequencing methods, like SMRT (Single Molecule, Real-Time) sequencing developed by Pacific Biosciences, offer much longer read length in comparison to Next Generation Sequencing (NGS) methods. Hence, they are well suited for de novo- or re-sequencing projects. Sequences generated for these purposes will not only contain reads originating from the nuclear genome, but also a significant amount of reads originating from the organelles of the target organism. These reads are usually discarded but they can also be used for an assembly of organellar replicons. The long read length supports resolution of repetitive regions and repeats within the organelles genome which might be problematic when just using short read data. Additionally, SMRT sequencing is less influenced by GC rich areas and by long stretches of the same base.

Results

We describe a workflow for a de novo assembly of the sugar beet (Beta vulgaris ssp. vulgaris) chloroplast genome sequence only based on data originating from a SMRT sequencing dataset targeted on its nuclear genome. We show that the data obtained from such an experiment are sufficient to create a high quality assembly with a higher reliability than assemblies derived from e.g. Illumina reads only. The chloroplast genome is especially challenging for de novo assembling as it contains two large inverted repeat (IR) regions. We also describe some limitations that still apply even though long reads are used for the assembly.

Conclusions

SMRT sequencing reads extracted from a dataset created for nuclear genome (re)sequencing can be used to obtain a high quality de novo assembly of the chloroplast of the sequenced organism. Even with a relatively small overall coverage for the nuclear genome it is possible to collect more than enough reads to generate a high quality assembly that outperforms short read based assemblies. However, even with long reads it is not always possible to clarify the order of elements of a chloroplast genome sequence reliantly which we could demonstrate with Fosmid End Sequences (FES) generated with Sanger technology. Nevertheless, this limitation also applies to short read sequencing data but is reached in this case at a much earlier stage during finishing.

Electronic supplementary material

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An improved genome of the model marine alga Ostreococcus tauri unfolds by assessing Illumina de novo assemblies

Background

Cost effective next generation sequencing technologies now enable the production of genomic datasets for many novel planktonic eukaryotes, representing an understudied reservoir of genetic diversity. O. tauri is the smallest free-living photosynthetic eukaryote known to date, a coccoid green alga that was first isolated in 1995 in a lagoon by the Mediterranean sea. Its simple features, ease of culture and the sequencing of its 13 Mb haploid nuclear genome have promoted this microalga as a new model organism for cell biology. Here, we investigated the quality of genome assemblies of Illumina GAIIx 75 bp paired-end reads from Ostreococcus tauri, thereby also improving the existing assembly and showing the genome to be stably maintained in culture.

Results

The 3 assemblers used, ABySS, CLCBio and Velvet, produced 95% complete genomes in 1402 to 2080 scaffolds with a very low rate of misassembly. Reciprocally, these assemblies improved the original genome assembly by filling in 930 gaps. Combined with additional analysis of raw reads and PCR sequencing effort, 1194 gaps have been solved in total adding up to 460 kb of sequence. Mapping of RNAseq Illumina data on this updated genome led to a twofold reduction in the proportion of multi-exon protein coding genes, representing 19% of the total 7699 protein coding genes. The comparison of the DNA extracted in 2001 and 2009 revealed the fixation of 8 single nucleotide substitutions and 2 deletions during the approximately 6000 generations in the lab. The deletions either knocked out or truncated two predicted transmembrane proteins, including a glutamate-receptor like gene.

Conclusion

High coverage (>80 fold) paired-end Illumina sequencing enables a high quality 95% complete genome assembly of a compact ~13 Mb haploid eukaryote. This genome sequence has remained stable for 6000 generations of lab culture.

Electronic supplementary material

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Read Length and Repeat Resolution: Exploring Prokaryote Genomes Using Next-Generation Sequencing Technologies

Background

There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats.

Methodology/Principal Findings

Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads.

Conclusions

Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length.     

Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma

Background

Oral squamous cell carcinoma (OSCC) is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA) mutations in primary oral tumors, recurrences and metastases.

Methods

We performed an in-depth validation of mtDNA next-generation sequencing (NGS) on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb) from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood) collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base).

Results

We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p<0.001). Non-synonymous heteroplasmic variants were enriched among cancerous tissues. The proportions of somatic and inherited variants in a given gene region were strongly correlated (r = 0.85; p<0.001). Half of the patients shared mutations between benign and cancerous tissue samples. Low level heteroplasmies (<10%) were more frequent in benign samples compared to tumor samples, where heteroplasmies >10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases.

Conclusions

We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer.     

Single molecule sequencing and genome assembly of a clinical specimen of Loa loa,the causative agent of loiasis

Background

More than 20% of the world’s population is at risk for infection by filarial nematodes and >180 million people worldwide are already infected. Along with infection comes significant morbidity that has a socioeconomic impact. The eight filarial nematodes that infect humans are Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, Loa loa, Mansonella perstans, Mansonella streptocerca, and Mansonella ozzardi, of which three have published draft genome sequences. Since all have humans as the definitive host, standard avenues of research that rely on culturing and genetics have often not been possible. Therefore, genome sequencing provides an important window into understanding the biology of these parasites. The need for large amounts of high quality genomic DNA from homozygous, inbred lines; the availability of only short sequence reads from next-generation sequencing platforms at a reasonable expense; and the lack of random large insert libraries has limited our ability to generate high quality genome sequences for these parasites. However, the Pacific Biosciences single molecule, real-time sequencing platform holds great promise in reducing input amounts and generating sufficiently long sequences that bypass the need for large insert paired libraries.

Results

Here, we report on efforts to generate a more complete genome assembly for L. loa using genetically heterogeneous DNA isolated from a single clinical sample and sequenced on the Pacific Biosciences platform. To obtain the best assembly, numerous assemblers and sequencing datasets were analyzed, combined, and compared. Quiver-informed trimming of an assembly of only Pacific Biosciences reads by HGAP2 was selected as the final assembly of 96.4 Mbp in 2,250 contigs. This results in ~9% more of the genome in ~85% fewer contigs from ~80% less starting material at a fraction of the cost of previous Roche 454-based sequencing efforts.

Conclusions

The result is the most complete filarial nematode assembly produced thus far and demonstrates the utility of single molecule sequencing on the Pacific Biosciences platform for genetically heterogeneous metazoan genomes.

Electronic supplementary material

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An improved genome reference for the African cichlid,Metriaclima zebra

Background

Problems associated with using draft genome assemblies are well documented and have become more pronounced with the use of short read data for de novo genome assembly. We set out to improve the draft genome assembly of the African cichlid fish, Metriaclima zebra, using a set of Pacific Biosciences SMRT sequencing reads corresponding to 16.5× coverage of the genome. Here we characterize the improvements that these long reads allowed us to make to the state-of-the-art draft genome previously assembled from short read data.

Results

Our new assembly closed 68 % of the existing gaps and added 90.6Mbp of new non-gap sequence to the existing draft assembly of M. zebra. Comparison of the new assembly to the sequence of several bacterial artificial chromosome clones confirmed the accuracy of the new assembly. The closure of sequence gaps revealed thousands of new exons, allowing significant improvement in gene models. We corrected one known misassembly, and identified and fixed other likely misassemblies. 63.5 Mbp (70 %) of the new sequence was classified as repetitive and the new sequence allowed for the assembly of many more transposable elements.

Conclusions

Our improvements to the M. zebra draft genome suggest that a reasonable investment in long reads could greatly improve many comparable vertebrate draft genome assemblies.

Electronic supplementary material

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Construction of a SNP-based genetic linkage map in cultivated peanut based on large scale marker development using next-generation double-digest restriction-site-associated DNA sequencing (ddRADseq)

Background

Cultivated peanut, or groundnut (Arachis hypogaea L.), is an important oilseed crop with an allotetraploid genome (AABB, 2n = 4x = 40). In recent years, many efforts have been made to construct linkage maps in cultivated peanut, but almost all of these maps were constructed using low-throughput molecular markers, and most show a low density, directly influencing the value of their applications. With advances in next-generation sequencing (NGS) technology, the construction of high-density genetic maps has become more achievable in a cost-effective and rapid manner. The objective of this study was to establish a high-density single nucleotide polymorphism (SNP)-based genetic map for cultivated peanut by analyzing next-generation double-digest restriction-site-associated DNA sequencing (ddRADseq) reads.

Results

We constructed reduced representation libraries (RRLs) for two A. hypogaea lines and 166 of their recombinant inbred line (RIL) progenies using the ddRADseq technique. Approximately 175 gigabases of data containing 952,679,665 paired-end reads were obtained following Solexa sequencing. Mining this dataset, 53,257 SNPs were detected between the parents, of which 14,663 SNPs were also detected in the population, and 1,765 of the obtained polymorphic markers met the requirements for use in the construction of a genetic map. Among 50 randomly selected in silico SNPs, 47 were able to be successfully validated. One linkage map was constructed, which was comprised of 1,685 marker loci, including 1,621 SNPs and 64 simple sequence repeat (SSR) markers. The map displayed a distribution of the markers into 20 linkage groups (LGs A01–A10 and B01–B10), spanning a distance of 1,446.7 cM. The alignment of the LGs from this map was shown in comparison with a previously integrated consensus map from peanut.

Conclusions

This study showed that the ddRAD library combined with NGS allowed the rapid discovery of a large number of SNPs in the cultivated peanut. The first high density SNP-based linkage map for A. hypogaea was generated that can serve as a reference map for cultivated Arachis species and will be useful in genetic mapping. Our results contribute to the available molecular marker resources and to the assembly of a reference genome sequence for the peanut.

Electronic supplementary material

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Whole Genome Amplification and De novo Assembly of Single Bacterial Cells

Background

Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells.

Methodology/Principal Findings

We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs.

Conclusions/Significance

The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.     

Efficient and accurate whole genome assembly and methylome profiling of E. coli

Background

With the price of next generation sequencing steadily decreasing, bacterial genome assembly is now accessible to a wide range of researchers. It is therefore necessary to understand the best methods for generating a genome assembly, specifically, which combination of sequencing and bioinformatics strategies result in the most accurate assemblies. Here, we sequence three E. coli strains on the Illumina MiSeq, Life Technologies Ion Torrent PGM, and Pacific Biosciences RS. We then perform genome assemblies on all three datasets alone or in combination to determine the best methods for the assembly of bacterial genomes.

Results

Three E. coli strains – BL21(DE3), Bal225, and DH5α – were sequenced to a depth of 100× on the MiSeq and Ion Torrent machines and to at least 125× on the PacBio RS. Four assembly methods were examined and compared. The previously published BL21(DE3) genome [GenBank:{"type":"entrez-nucleotide","attrs":{"text":"AM946981.2","term_id":"313848522","term_text":"AM946981.2"}}AM946981.2], allowed us to evaluate the accuracy of each of the BL21(DE3) assemblies. BL21(DE3) PacBio-only assemblies resulted in a 90% reduction in contigs versus short read only assemblies, while N50 numbers increased by over 7-fold. Strikingly, the number of SNPs in PacBio-only assemblies were less than half that seen with short read assemblies (~20 SNPs vs. ~50 SNPs) and indels also saw dramatic reductions (~2 indel >5 bp in PacBio-only assemblies vs. ~12 for short-read only assemblies). Assemblies that used a mixture of PacBio and short read data generally fell in between these two extremes. Use of PacBio sequencing reads also allowed us to call covalent base modifications for the three strains. Each of the strains used here had a known covalent base modification genotype, which was confirmed by PacBio sequencing.

Conclusion

Using data generated solely from the Pacific Biosciences RS, we were able to generate the most complete and accurate de novo assemblies of E. coli strains. We found that the addition of other sequencing technology data offered no improvements over use of PacBio data alone. In addition, the sequencing data from the PacBio RS allowed for sensitive and specific calling of covalent base modifications.

Electronic supplementary material

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Optimizing Information in Next-Generation-Sequencing (NGS) Reads for Improving De Novo Genome Assembly

Next-Generation-Sequencing is advantageous because of its much higher data throughput and much lower cost compared with the traditional Sanger method. However, NGS reads are shorter than Sanger reads, making de novo genome assembly very challenging. Because genome assembly is essential for all downstream biological studies, great efforts have been made to enhance the completeness of genome assembly, which requires the presence of long reads or long distance information. To improve de novo genome assembly, we develop a computational program, ARF-PE, to increase the length of Illumina reads. ARF-PE takes as input Illumina paired-end (PE) reads and recovers the original DNA fragments from which two ends the paired reads are obtained. On the PE data of four bacteria, ARF-PE recovered >87% of the DNA fragments and achieved >98% of perfect DNA fragment recovery. Using Velvet, SOAPdenovo, Newbler, and CABOG, we evaluated the benefits of recovered DNA fragments to genome assembly. For all four bacteria, the recovered DNA fragments increased the assembly contiguity. For example, the N50 lengths of the P. brasiliensis contigs assembled by SOAPdenovo and Newbler increased from 80,524 bp to 166,573 bp and from 80,655 bp to 193,388 bp, respectively. ARF-PE also increased assembly accuracy in many cases. On the PE data of two fungi and a human chromosome, ARF-PE doubled and tripled the N50 length. However, the assembly accuracies dropped, but still remained >91%. In general, ARF-PE can increase both assembly contiguity and accuracy for bacterial genomes. For complex eukaryotic genomes, ARF-PE is promising because it raises assembly contiguity. But future error correction is needed for ARF-PE to also increase the assembly accuracy. ARF-PE is freely available at http://140.116.235.124/~tliu/arf-pe/.     

A Pathogenic Mosaic TP53 Mutation in Two Germ Layers Detected by Next Generation Sequencing

Background

Li-Fraumeni syndrome is caused by germline TP53 mutations and is clinically characterized by a predisposition to a range of cancers, most commonly sarcoma, brain tumours and leukemia. Pathogenic mosaic TP53 mutations have only rarely been described.

Methods and Findings

We describe a 2 years old child presenting with three separate cancers over a 6 month period; two soft tissue mesenchymal tumors and an aggressive metastatic neuroblastoma. As conventional testing of blood DNA by Sanger sequencing for mutations in TP53, ALK, and SDH was negative, whole exome sequencing of the blood DNA of the patient and both parents was performed to screen more widely for cancer predisposing mutations. In the patient''s but not the parents'' DNA we found a c.743 G>A, p.Arg248Gln (CCDS11118.1) TP53 mutation in 3–20% of sequencing reads, a level that would not generally be detectable by Sanger sequencing. Homozygosity for this mutation was detected in all tumor samples analyzed, and germline mosaicism was demonstrated by analysis of the child''s newborn blood spot DNA. The occurrence of separate tumors derived from different germ layers suggests that this de novo mutation occurred early in embryogenesis, prior to gastrulation.

Conclusion

The case demonstrates pathogenic mosaicim, detected by next generation deep sequencing, that arose in the early stages of embryogenesis.     

The Feasibility Study of Non-Invasive Fetal Trisomy 18 and 21 Detection with Semiconductor Sequencing Platform

Objective

Recent non-invasive prenatal testing (NIPT) technologies are based on next-generation sequencing (NGS). NGS allows rapid and effective clinical diagnoses to be determined with two common sequencing systems: Illumina and Ion Torrent platforms. The majority of NIPT technology is associated with Illumina platform. We investigated whether fetal trisomy 18 and 21 were sensitively and specifically detectable by semiconductor sequencer: Ion Proton.

Methods

From March 2012 to October 2013, we enrolled 155 pregnant women with fetuses who were diagnosed as high risk of fetal defects at Xiamen Maternal & Child Health Care Hospital (Xiamen, Fujian, China). Adapter-ligated DNA libraries were analyzed by the Ion Proton™ System (Life Technologies, Grand Island, NY, USA) with an average 0.3× sequencing coverage per nucleotide. Average total raw reads per sample was 6.5 million and mean rate of uniquely mapped reads was 59.0%. The results of this study were derived from BWA mapping. Z-score was used for fetal trisomy 18 and 21 detection.

Results

Interactive dot diagrams showed the minimal z-score values to discriminate negative versus positive cases of fetal trisomy 18 and 21. For fetal trisomy 18, the minimal z-score value of 2.459 showed 100% positive predictive and negative predictive values. The minimal z-score of 2.566 was used to classify negative versus positive cases of fetal trisomy 21.

Conclusion

These results provide the evidence that fetal trisomy 18 and 21 detection can be performed with semiconductor sequencer. Our data also suggest that a prospective study should be performed with a larger cohort of clinically diverse obstetrics patients.     

Mapping Accuracy of Short Reads from Massively Parallel Sequencing and the Implications for Quantitative Expression Profiling

Background

Massively parallel sequencing offers an enormous potential for expression profiling, in particular for interspecific comparisons. Currently, different platforms for massively parallel sequencing are available, which differ in read length and sequencing costs. The 454-technology offers the highest read length. The other sequencing technologies are more cost effective, on the expense of shorter reads. Reliable expression profiling by massively parallel sequencing depends crucially on the accuracy to which the reads could be mapped to the corresponding genes.

Methodology/Principal Findings

We performed an in silico analysis to evaluate whether incorrect mapping of the sequence reads results in a biased expression pattern. A comparison of six available mapping software tools indicated a considerable heterogeneity in mapping speed and accuracy. Independently of the software used to map the reads, we found that for compact genomes both short (35 bp, 50 bp) and long sequence reads (100 bp) result in an almost unbiased expression pattern. In contrast, for species with a larger genome containing more gene families and repetitive DNA, shorter reads (35–50 bp) produced a considerable bias in gene expression. In humans, about 10% of the genes had fewer than 50% of the sequence reads correctly mapped. Sequence polymorphism up to 9% had almost no effect on the mapping accuracy of 100 bp reads. For 35 bp reads up to 3% sequence divergence did not affect the mapping accuracy strongly. The effect of indels on the mapping efficiency strongly depends on the mapping software.

Conclusions/Significance

In complex genomes, expression profiling by massively parallel sequencing could introduce a considerable bias due to incorrectly mapped sequence reads if the read length is short. Nevertheless, this bias could be accounted for if the genomic sequence is known. Furthermore, sequence polymorphisms and indels also affect the mapping accuracy and may cause a biased gene expression measurement. The choice of the mapping software is highly critical and the reliability depends on the presence/absence of indels and the divergence between reads and the reference genome. Overall, we found SSAHA2 and CLC to produce the most reliable mapping results.