Design considerations for massively parallel sequencing studies of complex human disease

Massively Parallel Sequencing (MPS) allows sequencing of entire exomes and genomes to now be done at reasonable cost, and its utility for identifying genes responsible for rare Mendelian disorders has been demonstrated. However, for a complex disease, study designs need to accommodate substantial degrees of locus, allelic, and phenotypic heterogeneity, as well as complex relationships between genotype and phenotype. Such considerations include careful selection of samples for sequencing and a well-developed strategy for identifying the few "true" disease susceptibility genes from among the many irrelevant genes that will be found to harbor rare variants. To examine these issues we have performed simulation-based analyses in order to compare several strategies for MPS sequencing in complex disease. Factors examined include genetic architecture, sample size, number and relationship of individuals selected for sequencing, and a variety of filters based on variant type, multiple observations of genes and concordance of genetic variants within pedigrees. A two-stage design was assumed where genes from the MPS analysis of high-risk families are evaluated in a secondary screening phase of a larger set of probands with more modest family histories. Designs were evaluated using a cost function that assumes the cost of sequencing the whole exome is 400 times that of sequencing a single candidate gene. Results indicate that while requiring variants to be identified in multiple pedigrees and/or in multiple individuals in the same pedigree are effective strategies for reducing false positives, there is a danger of over-filtering so that most true susceptibility genes are missed. In most cases, sequencing more than two individuals per pedigree results in reduced power without any benefit in terms of reduced overall cost. Further, our results suggest that although no single strategy is optimal, simulations can provide important guidelines for study design.     

Review of massively parallel DNA sequencing technologies

Since the development of technologies that can determine the base-pair sequence of DNA, the ability to sequence genes has contributed much to science and medicine. However, it has remained a relatively costly and laborious process, hindering its use as a routine biomedical tool. Recent times are seeing rapid developments in this field, both in the availability of novel sequencing platforms, as well as supporting technologies involved in processes such as targeting and data analysis. This is leading to significant reductions in the cost of sequencing a human genome and the potential for its use as a routine biomedical tool. This review is a snapshot of this rapidly moving field examining the current state of the art, forthcoming developments and some of the issues still to be resolved prior to the use of new sequencing technologies in routine clinical diagnosis.     

Targeted single molecule mutation detection with massively parallel sequencing

Next-generation sequencing (NGS) technologies have transformed genomic research and have the potential to revolutionize clinical medicine. However, the background error rates of sequencing instruments and limitations in targeted read coverage have precluded the detection of rare DNA sequence variants by NGS. Here we describe a method, termed CypherSeq, which combines double-stranded barcoding error correction and rolling circle amplification (RCA)-based target enrichment to vastly improve NGS-based rare variant detection. The CypherSeq methodology involves the ligation of sample DNA into circular vectors, which contain double-stranded barcodes for computational error correction and adapters for library preparation and sequencing. CypherSeq is capable of detecting rare mutations genome-wide as well as those within specific target genes via RCA-based enrichment. We demonstrate that CypherSeq is capable of correcting errors incurred during library preparation and sequencing to reproducibly detect mutations down to a frequency of 2.4 × 10⁻⁷ per base pair, and report the frequency and spectra of spontaneous and ethyl methanesulfonate-induced mutations across the Saccharomycescerevisiae genome.     

Comprehensive microRNA profiling in B-cells of human centenarians by massively parallel sequencing

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and play a critical role in development, homeostasis, and disease. Despite their demonstrated roles in age-associated pathologies, little is known about the role of miRNAs in human aging and longevity. RESULTS: We employed massively parallel sequencing technology to identify miRNAs expressed in B-cells from Ashkenazi Jewish centenarians, i.e., those living to a hundred and a human model of exceptional longevity, and younger controls without a family history of longevity. With data from 26.7 million reads comprising 9.4x108 bp from 3 centenarian and 3 control individuals, we discovered a total of 276 known miRNAs and 8 unknown miRNAs ranging several orders of magnitude in expression levels, a typical characteristics of saturated miRNA-sequencing. A total of 22 miRNAs were found to be significantly upregulated, with only 2 miRNAs downregulated, in centenarians as compared to controls. Gene Ontology analysis of the predicted and validated targets of the 24 differentially expressed miRNAs indicated enrichment of functional pathways involved in cell metabolism, cell cycle, cell signaling, and cell differentiation. A cross sectional expression analysis of the differentially expressed miRNAs in B-cells from Ashkenazi Jewish individuals between the 50th and 100th years of age indicated that expression levels of miR-363* declined significantly with age. Centenarians, however, maintained the youthful expression level. This result suggests that miR-363* may be a candidate longevity-associated miRNA. CONCLUSION: Our comprehensive miRNA data provide a resource for further studies to identify genetic pathways associated with aging and longevity in humans.     

Flow cytometry for enrichment and titration in massively parallel DNA sequencing

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols.     

Cross-platform compatibility of Hi-Plex,a streamlined approach for targeted massively parallel sequencing

Although per-base sequencing costs have decreased during recent years, library preparation for targeted massively parallel sequencing remains constrained by high reagent cost, limited design flexibility, and protocol complexity. To address these limitations, we previously developed Hi-Plex, a polymerase chain reaction (PCR) massively parallel sequencing strategy for screening panels of genomic target regions. Here, we demonstrate that Hi-Plex applied with hybrid adapters can generate a library suitable for sequencing with both the Ion Torrent and the TruSeq chemistries and that adjusting primer concentrations improves coverage uniformity. These results expand Hi-Plex capabilities as an accurate, affordable, flexible, and rapid approach for various genetic screening applications.     

Non-invasive prenatal diagnosis by massively parallel sequencing of maternal plasma DNA

The presence of foetal DNA in the plasma of pregnant women has opened up new possibilities for non-invasive prenatal diagnosis. The use of circulating foetal DNA for the non-invasive prenatal detection of foetal chromosomal aneuploidies is challenging as foetal DNA represents a minor fraction of maternal plasma DNA. In 2007, it was shown that single molecule counting methods would allow the detection of the presence of a trisomic foetus, as long as enough molecules were counted. With the advent of massively parallel sequencing, millions or billions of DNA molecules can be readily counted. Using massively parallel sequencing, foetal trisomies 21, 13 and 18 have been detected from maternal plasma. Recently, large-scale clinical studies have validated the robustness of this approach for the prenatal detection of foetal chromosomal aneuploidies. A proof-of-concept study has also shown that a genome-wide genetic and mutational map of a foetus can be constructed from the maternal plasma DNA sequencing data. These developments suggest that the analysis of foetal DNA in maternal plasma would play an increasingly important role in future obstetrics practice. It is thus a priority that the ethical, social and legal issues regarding this technology be systematically studied.     

Prenatal detection of aneuploidy and imbalanced chromosomal arrangements by massively parallel sequencing

Fetal chromosomal abnormalities are the most common reasons for invasive prenatal testing. Currently, G-band karyotyping and several molecular genetic methods have been established for diagnosis of chromosomal abnormalities. Although these testing methods are highly reliable, the major limitation remains restricted resolutions or can only achieve limited coverage on the human genome at one time. The massively parallel sequencing (MPS) technologies which can reach single base pair resolution allows detection of genome-wide intragenic deletions and duplication challenging karyotyping and microarrays as the tool for prenatal diagnosis. Here we reported a novel and robust MPS-based method to detect aneuploidy and imbalanced chromosomal arrangements in amniotic fluid (AF) samples. We sequenced 62 AF samples on Illumina GAIIx platform and with averagely 0.01× whole genome sequencing data we detected 13 samples with numerical chromosomal abnormalities by z-test. With up to 2× whole genome sequencing data we were able to detect microdeletion/microduplication (ranged from 1.4 Mb to 37.3 Mb of 5 samples from chorionic villus sampling (CVS) using SeqSeq algorithm. Our work demonstrated MPS is a robust and accurate approach to detect aneuploidy and imbalanced chromosomal arrangements in prenatal samples.     

A viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing

Here we describe a virus discovery protocol for a range of different virus genera, that can be applied to biopsy-sized tissue samples. Our viral enrichment procedure, validated using canine and human liver samples, significantly improves viral read copy number and increases the length of viral contigs that can be generated by de novo assembly. This in turn enables the Illumina next generation sequencing (NGS) platform to be used as an effective tool for viral discovery from tissue samples.     

FAST-SeqS: a simple and efficient method for the detection of aneuploidy by massively parallel sequencing

Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair designed to amplify a discrete subset of repeated regions. Using this approach, samples containing as little as 4% trisomy 21 DNA could be readily distinguished from euploid samples.     

Genome analysis of the domestic dog (Korean Jindo) by massively parallel sequencing

Although pioneering sequencing projects have shed light on the boxer and poodle genomes, a number of challenges need to be met before the sequencing and annotation of the dog genome can be considered complete. Here, we present the DNA sequence of the Jindo dog genome, sequenced to 45-fold average coverage using Illumina massively parallel sequencing technology. A comparison of the sequence to the reference boxer genome led to the identification of 4 675 437 single nucleotide polymorphisms (SNPs, including 3 346 058 novel SNPs), 71 642 indels and 8131 structural variations. Of these, 339 non-synonymous SNPs and 3 indels are located within coding sequences (CDS). In particular, 3 non-synonymous SNPs and a 26-bp deletion occur in the TCOF1 locus, implying that the difference observed in cranial facial morphology between Jindo and boxer dogs might be influenced by those variations. Through the annotation of the Jindo olfactory receptor gene family, we found 2 unique olfactory receptor genes and 236 olfactory receptor genes harbouring non-synonymous homozygous SNPs that are likely to affect smelling capability. In addition, we determined the DNA sequence of the Jindo dog mitochondrial genome and identified Jindo dog-specific mtDNA genotypes. This Jindo genome data upgrade our understanding of dog genomic architecture and will be a very valuable resource for investigating not only dog genetics and genomics but also human and dog disease genetics and comparative genomics.     

Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes

Background

Mycoheterotrophic plants are considered to associate very specifically with fungi. Mycoheterotrophic orchids are mostly associated with ectomycorrhizal fungi in temperate regions, or with saprobes or parasites in tropical regions. Although most mycoheterotrophic orchids occur in the tropics, few studies have been devoted to them, and the main conclusions about their specificity have hitherto been drawn from their association with ectomycorrhizal fungi in temperate regions.

Results

We investigated three Asiatic Neottieae species from ectomycorrhizal forests in Thailand. We found that all were associated with ectomycorrhizal fungi, such as Thelephoraceae, Russulaceae and Sebacinales. Based on ¹³C enrichment of their biomass, they probably received their organic carbon from these fungi, as do mycoheterotrophic Neottieae from temperate regions. Moreover, ¹³C enrichment suggested that some nearby green orchids received part of their carbon from fungi too. Nevertheless, two of the three orchids presented a unique feature for mycoheterotrophic plants: they were not specifically associated with a narrow clade of fungi. Some orchid individuals were even associated with up to nine different fungi.

Conclusion

Our results demonstrate that some green and mycoheterotrophic orchids in tropical regions can receive carbon from ectomycorrhizal fungi, and thus from trees. Our results reveal the absence of specificity in two mycoheterotrophic orchid-fungus associations in tropical regions, in contrast to most previous studies of mycoheterotrophic plants, which have been mainly focused on temperate orchids.     

Water mass-specificity of bacterial communities in the North Atlantic revealed by massively parallel sequencing

Bacterial assemblages from subsurface (100 m depth), meso- (200-1000 m depth) and bathy-pelagic (below 1000 m depth) zones at 10 stations along a North Atlantic Ocean transect from 60°N to 5°S were characterized using massively parallel pyrotag sequencing of the V6 region of the 16S rRNA gene (V6 pyrotags). In a dataset of more than 830,000 pyrotags, we identified 10,780 OTUs of which 52% were singletons. The singletons accounted for less than 2% of the OTU abundance, whereas the 100 and 1000 most abundant OTUs represented 80% and 96% respectively of all recovered OTUs. Non-metric Multi-Dimensional Scaling and Canonical Correspondence Analysis of all the OTUs excluding the singletons revealed a clear clustering of the bacterial communities according to the water masses. More than 80% of the 1000 most abundant OTUs corresponded to Proteobacteria of which 55% were Alphaproteobacteria, mostly composed of the SAR11 cluster. Gammaproteobacteria increased with depth and included a relatively large number of OTUs belonging to Alteromonadales and Oceanospirillales. The bathypelagic zone showed higher taxonomic evenness than the overlying waters, albeit bacterial diversity was remarkably variable. Both abundant and low-abundance OTUs were responsible for the distinct bacterial communities characterizing the major deep-water masses. Taken together, our results reveal that deep-water masses act as bio-oceanographic islands for bacterioplankton leading to water mass-specific bacterial communities in the deep waters of the Atlantic.     

BreaKmer: detection of structural variation in targeted massively parallel sequencing data using kmers

Genomic structural variation (SV), a common hallmark of cancer, has important predictive and therapeutic implications. However, accurately detecting SV using high-throughput sequencing data remains challenging, especially for ‘targeted’ resequencing efforts. This is critically important in the clinical setting where targeted resequencing is frequently being applied to rapidly assess clinically actionable mutations in tumor biopsies in a cost-effective manner. We present BreaKmer, a novel approach that uses a ‘kmer’ strategy to assemble misaligned sequence reads for predicting insertions, deletions, inversions, tandem duplications and translocations at base-pair resolution in targeted resequencing data. Variants are predicted by realigning an assembled consensus sequence created from sequence reads that were abnormally aligned to the reference genome. Using targeted resequencing data from tumor specimens with orthogonally validated SV, non-tumor samples and whole-genome sequencing data, BreaKmer had a 97.4% overall sensitivity for known events and predicted 17 positively validated, novel variants. Relative to four publically available algorithms, BreaKmer detected SV with increased sensitivity and limited calls in non-tumor samples, key features for variant analysis of tumor specimens in both the clinical and research settings.