SESAME (SEquence Sorter & AMplicon Explorer): genotyping based on high-throughput multiplex amplicon sequencing

SUMMARY: Characterizing genetic diversity through genotyping short amplicons is central to evolutionary biology. Next-generation sequencing (NGS) technologies changed the scale at which these type of data are acquired. SESAME is a web application package that assists genotyping of multiplexed individuals for several markers based on NGS amplicon sequencing. It automatically assigns reads to loci and individuals, corrects reads if standard samples are available and provides an intuitive graphical user interface (GUI) for allele validation based on the sequences and associated decision-making tools. The aim of SESAME is to help allele identification among a large number of sequences. AVAILABILITY: SESAME and its documentation are freely available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported Licence for Windows and Linux from http://www1.montpellier.inra.fr/CBGP/NGS/ or http://tinyurl.com/ngs-sesame.     

Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes

Background

PCR amplicon sequencing has been widely used as a targeted approach for both DNA and RNA sequence analysis. High multiplex PCR has further enabled the enrichment of hundreds of amplicons in one simple reaction. At the same time, the performance of PCR amplicon sequencing can be negatively affected by issues such as high duplicate reads, polymerase artifacts and PCR amplification bias. Recently researchers have made some good progress in addressing these shortcomings by incorporating molecular barcodes into PCR primer design. So far, most work has been demonstrated using one to a few pairs of primers, which limits the size of the region one can analyze.

Results

We developed a simple protocol, which enables the use of molecular barcodes in high multiplex PCR with hundreds of amplicons. Using this protocol and reference materials, we demonstrated the applications in accurate variant calling at very low fraction over a large region and in targeted RNA quantification. We also evaluated the protocol’s utility in profiling FFPE samples.

Conclusions

We demonstrated the successful implementation of molecular barcodes in high multiplex PCR, with multiplex scale many times higher than earlier work. We showed that the new protocol combines the benefits of both high multiplex PCR and molecular barcodes, i.e. the analysis of a very large region, low DNA input requirement, very good reproducibility and the ability to detect as low as 1 % mutations with minimal false positives (FP).

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1806-8) contains supplementary material, which is available to authorized users.     

Identification of genome-wide single nucleotide polymorphisms in allopolyploid crop Brassica napus

Background

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay.

Results

A total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%.

Conclusions

Despite the polyploid nature of B. napus, nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.     

A high-throughput SNP array in the amphidiploid species Brassica napus shows diversity in resistance genes

Single-nucleotide polymorphisms (SNPs)are molecular markers based on nucleotide variation and can be used for genotyping assays across populations and to track genomic inheritance. SNPs offer a comprehensive genotyping alternative to whole-genome sequencing for both agricultural and research purposes including molecular breeding and diagnostics, genome evolution and genetic diversity analyses, genetic mapping, and trait association studies. Here genomic SNPs were discovered between four cultivars of the important amphidiploid oilseed species Brassica napus and used to develop a B. napus Infinium? array containing 5,306 SNPs randomly dispersed across the genome. Assay success was high, with >94 % of these producing a reproducible, polymorphic genotype in the 1,070 samples screened. Although the assay was designed to B. napus, successful SNP amplification was achieved in the B. napus progenitor species, Brassica rapa and Brassica oleracea, and to a lesser extent in the related species Brassica nigra. Phylogenetic analysis was consistent with the expected relationships between B. napus individuals. This study presents an efficient custom SNP assay development pipeline in the complex polyploid Brassica genome and demonstrates the utility of the array for high-throughput genotyping in a number of related Brassica species. It also demonstrates the utility of this assay in genotyping resistance genes on chromosome A7, which segregate amongst the 1,070 samples.     

Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

ABSTRACT: BACKGROUND: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. RESULTS: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. CONCLUSIONS: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.     

Ordered catenation of sequence-tagged sites and multiplexed SNP genotyping by sequencing

We describe a method for the efficient genotyping of SNPs, involving sequencing of ordered and catenated sequence-tagged sites (OCS). In OCS, short genomic segments, each containing an SNP, are amplified by PCR using primers that carry specially designed extra nucleotides at their 5′-ends. Amplification products are then combined and converted to a concatamer in a defined order by a second round of thermal cycling. The concatenation takes place because the 5′-ends of each amplicon are designed to be complementary to the ends of the presumptive neighboring amplicons. The primer sequences for OCS are chosen using newly developed dedicated software, OCS Optimizer. Using sets of SNPs, we show that at least 10 STSs can be concatenated in a predefined order and all SNPs in the STSs are accurately genotyped by one two-way sequencing reaction.     

High-throughput multiplex HLA genotyping by next-generation sequencing using multi-locus individual tagging

Background

Unambiguous human leukocyte antigen (HLA) typing is important in transplant matching and disease association studies. High-resolution HLA typing that is not restricted to the peptide-binding region can decrease HLA allele ambiguities. Cost and technology constraints have hampered high-throughput and efficient high resolution unambiguous HLA typing. We have developed a method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency. Unambiguous HLA-A, B, C and DRB1 genotypes can be determined for 96 individuals in a single run of the Illumina MiSeq.

Results

Long-range amplification of full-length HLA genes from four loci was performed in separate polymerase chain reactions (PCR) using primers and PCR conditions that were optimized to reduce co-amplification of other HLA loci. Amplicons from the four HLA loci of each individual were then pooled and subjected to enzymatic library generation. All four loci of an individual were then tagged with one unique index combination. This multi-locus individual tagging (MIT) method combined with NGS enabled the four loci of 96 individuals to be analyzed in a single 500 cycle sequencing paired-end run of the Illumina-MiSeq. The MIT-NGS method generated sequence reads from the four loci were then discriminated using commercially available NGS HLA typing software. Comparison of the MIT-NGS with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method.

Conclusions

The MIT-NGS method enabled accurate, robust and cost effective simultaneous analyses of four HLA loci per sample and produced 6 or 8-digit high-resolution unambiguous phased HLA typing data from 96 individuals in a single NGS run.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-864) contains supplementary material, which is available to authorized users.     

SNP genotyping through the melting analysis of unlabelled oligonucleotide applied on dilute PCR amplicon

Adaptation of DNA melting analysis for polymorphic single nucleotides (SNPs) genotyping using an unlabeled oligonucleotide probe for polymorphic DNAs under the presence of fluorescent DNA binding dye necessitates a reaction condition where the probe efficiently associates with a target strand that is PCR amplified. We present experimental evidence that application of an unlabeled probe to a dilute PCR amplicon provides a condition such that the fluorescent signals gained subsequently by probe melting are sufficient to discriminate allelic identities. This approach is best exploited by adapting the multiplexing PCR technique in order to cover multiple SNPs for given samples. 3′-end modification of the probe is unnecessary as the amplicon dilution step provides a way of inactivating the polymerase through divalent cation chelation. With the use of low-cost reagents and ordinary laboratory equipment, this method offers a rapid, simple and cost-efficient way of SNP genotyping.     

Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors

Investigating recombination of homoeologous chromosomes in allopolyploid species is central to understanding plant breeding and evolution. However, examining chromosome pairing in the allotetraploid Brassica napus has been hampered by the lack of chromosome-specific molecular probes. In this study, we establish the identification of all homoeologous chromosomes of allopolyploid B. napus by using robust molecular cytogenetic karyotypes developed for the progenitor species Brassica rapa (A genome) and Brassica oleracea (C genome). The identification of every chromosome among these three Brassica species utilized genetically mapped bacterial artificial chromosomes (BACs) from B. rapa as probes for fluorescent in situ hybridization (FISH). With this BAC-FISH data, a second karyotype was developed using two BACs that contained repetitive DNA sequences and the ubiquitous ribosomal and pericentromere repeats. Using this diagnostic probe mix and a BAC that contained a C-genome repeat in two successive hybridizations allowed for routine identification of the corresponding homoeologous chromosomes between the A and C genomes of B. napus. When applied to the B. napus cultivar Stellar, we detected one chromosomal rearrangement relative to the parental karyotypes. This robust novel chromosomal painting technique will have biological applications for the understanding of chromosome pairing, homoeologous recombination, and genome evolution in the genus Brassica and will facilitate new applied breeding technologies that rely upon identification of chromosomes.     

Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species

The ability to efficiently and accurately determine genotypes is a keystone technology in modern genetics, crucial to studies ranging from clinical diagnostics, to genotype-phenotype association, to reconstruction of ancestry and the detection of selection. To date, high capacity, low cost genotyping has been largely achieved via "SNP chip" microarray-based platforms which require substantial prior knowledge of both genome sequence and variability, and once designed are suitable only for those targeted variable nucleotide sites. This method introduces substantial ascertainment bias and inherently precludes detection of rare or population-specific variants, a major source of information for both population history and genotype-phenotype association. Recent developments in reduced-representation genome sequencing experiments on massively parallel sequencers (commonly referred to as RAD-tag or RADseq) have brought direct sequencing to the problem of population genotyping, but increased cost and procedural and analytical complexity have limited their widespread adoption. Here, we describe a complete laboratory protocol, including a custom combinatorial indexing method, and accompanying software tools to facilitate genotyping across large numbers (hundreds or more) of individuals for a range of markers (hundreds to hundreds of thousands). Our method requires no prior genomic knowledge and achieves per-site and per-individual costs below that of current SNP chip technology, while requiring similar hands-on time investment, comparable amounts of input DNA, and downstream analysis times on the order of hours. Finally, we provide empirical results from the application of this method to both genotyping in a laboratory cross and in wild populations. Because of its flexibility, this modified RADseq approach promises to be applicable to a diversity of biological questions in a wide range of organisms.     

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.     

Simultaneous discovery and testing of deletions for disease association in SNP genotyping studies

Copy-number variation (CNV), and deletions in particular, can play a crucial, causative role in rare disorders. The extent to which CNV contributes to common, complex disease etiology, however, is largely unknown. Current techniques to detect CNV are relatively expensive and time consuming, making it difficult to conduct the necessary large-scale genetic studies. SNP genotyping technologies, on the other hand, are relatively cheap, thereby facilitating large study designs. We have developed a computational tool capable of harnessing the information in SNP genotype data to detect deletions. Our approach not only detects deletions with high power but also returns accurate estimates of both the population frequency and the transmission frequency. This tool, therefore, lends itself to the discovery of deletions in large familial SNP genotype data sets and to simultaneous testing of the discovered deletion for association, with the use of both frequency-based and transmission/disequilibrium test-based designs. We demonstrate the effectiveness of our computer program (microdel), available for download at no cost, with both simulated and real data. Here, we report 693 deletions in the HapMap 16c collection, with each deletion assigned a population frequency.     

SNPstream UHT: ultra-high throughput SNP genotyping for pharmacogenomics and drug discovery

Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orrhid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-IT tag array single base extension genotyping technology The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHTArray Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1,000,000 genotyping assays with >96% of samples giving genotypes with >99% accuracy     

SNP discovery based on CATS and genotyping in the finless porpoise (Neophocaena phocaenoides)

Single nucleotide polymorphisms (SNPs) discovery and genotyping were performed for the finless porpoises (Neophocaena phocaenoides). About 202 comparative anchor tagged sequence primers derived from genomes of human, mouse and some other mammals were used to screen the finless porpoise population. Of the 51 SNPs discovered, 25 were further characterized with ideal genotyping primers and using fragment length discrepant allele specific PCR assay. This is the first report of SNP loci for the finless porpoise, which is helpful to provide some novel molecular markers and new genetic information relevant to the conservation and management of this endangered species.     

Genome- wide structural and functional variant discovery of rice landraces using genotyping by sequencing

Molecular Biology Reports - Rice landraces are vital genetic resources for agronomic and quality traits but the undeniable collection of Kerala landraces remains poorly delineated. To effectively...