TempliPhi: A Sequencing Template Preparation Procedure That Eliminates Overnight Cultures and DNA Purification

Preparing plasmid templates for DNA sequencing is the most time-consuming step in the sequencing process. Current template preparation methods rely on a labor-intensive, multistep procedure that takes up to 24 h and produces templates of varying quality and quantity. The TempliPhi™ DNA Sequencing Template Amplification Kit eliminates the requirement for extended bacterial growth prior to sequencing and saves laboratory personnel hands-on time by eliminating the centrifugation and transfer steps currently required by older preparatory methods. In addition, costly purification filters and columns are not necessary, as amplified product can be added directly to a sequencing reaction. Starting material can be any circular template from a colony, culture, glycerol stock, or plaque. Based on rolling circle amplification and employing bacteriophage Phi29 DNA polymerase, the method can produce 3–5 μg of template directly from a single bacterial colony in as little as 4 h. Implementation of these procedures in a laboratory or core sequencing facility can decrease cost on tips, plates, and other plasticware, while at the same time increase throughput.     

A Bayesian Framework to Identify Methylcytosines from High-Throughput Bisulfite Sequencing Data

High-throughput bisulfite sequencing technologies have provided a comprehensive and well-fitted way to investigate DNA methylation at single-base resolution. However, there are substantial bioinformatic challenges to distinguish precisely methylcytosines from unconverted cytosines based on bisulfite sequencing data. The challenges arise, at least in part, from cell heterozygosis caused by multicellular sequencing and the still limited number of statistical methods that are available for methylcytosine calling based on bisulfite sequencing data. Here, we present an algorithm, termed Bycom, a new Bayesian model that can perform methylcytosine calling with high accuracy. Bycom considers cell heterozygosis along with sequencing errors and bisulfite conversion efficiency to improve calling accuracy. Bycom performance was compared with the performance of Lister, the method most widely used to identify methylcytosines from bisulfite sequencing data. The results showed that the performance of Bycom was better than that of Lister for data with high methylation levels. Bycom also showed higher sensitivity and specificity for low methylation level samples (<1%) than Lister. A validation experiment based on reduced representation bisulfite sequencing data suggested that Bycom had a false positive rate of about 4% while maintaining an accuracy of close to 94%. This study demonstrated that Bycom had a low false calling rate at any methylation level and accurate methylcytosine calling at high methylation levels. Bycom will contribute significantly to studies aimed at recalibrating the methylation level of genomic regions based on the presence of methylcytosines.     

Production of random DNA oligomers for scalable DNA computing

While remarkably complex networks of connected DNA molecules can form from a relatively small number of distinct oligomer strands, a large computational space created by DNA reactions would ultimately require the use of many distinct DNA strands. The automatic synthesis of this many distinct strands is economically prohibitive. We present here a new approach to producing distinct DNA oligomers based on the polymerase chain reaction (PCR) amplification of a few random template sequences. As an example, we designed a DNA template sequence consisting of a 50-mer random DNA segment flanked by two 20-mer invariant primer sequences. Amplification of a dilute sample containing about 30 different template molecules allows us to obtain around 10¹¹ copies of these molecules and their complements. We demonstrate the use of these amplicons to implement some of the vector operations that will be required in a DNA implementation of an analog neural network.     

Degenerate adaptor sequences for detecting PCR duplicates in reduced representation sequencing data improve genotype calling accuracy

RAD‐tag is a powerful tool for high‐throughput genotyping. It relies on PCR amplification of the starting material, following enzymatic digestion and sequencing adaptor ligation. Amplification introduces duplicate reads into the data, which arise from the same template molecule and are statistically nonindependent, potentially introducing errors into genotype calling. In shotgun sequencing, data duplicates are removed by filtering reads starting at the same position in the alignment. However, restriction enzymes target specific locations within the genome, causing reads to start in the same place, and making it difficult to estimate the extent of PCR duplication. Here, we introduce a slight change to the Illumina sequencing adaptor chemistry, appending a unique four‐base tag to the first index read, which allows duplicate discrimination in aligned data. This approach was validated on the Illumina MiSeq platform, using double‐digest libraries of ants (Wasmannia auropunctata) and yeast (Saccharomyces cerevisiae) with known genotypes, producing modest though statistically significant gains in the odds of calling a genotype accurately. More importantly, removing duplicates also corrected for strong sample‐to‐sample variability of genotype calling accuracy seen in the ant samples. For libraries prepared from low‐input degraded museum bird samples (Mixornis gularis), which had low complexity, having been generated from relatively few starting molecules, adaptor tags show that virtually all of the genotypes were called with inflated confidence as a result of PCR duplicates. Quantification of library complexity by adaptor tagging does not significantly increase the difficulty of the overall workflow or its cost, but corrects for differences in quality between samples and permits analysis of low‐input material.     

Genome Wide Sampling Sequencing for SNP Genotyping: Methods,Challenges and Future Development

Genetic polymorphisms, particularly single nucleotide polymorphisms (SNPs), have been widely used to advance quantitative, functional and evolutionary genomics. Ideally, all genetic variants among individuals should be discovered when next generation sequencing (NGS) technologies and platforms are used for whole genome sequencing or resequencing. In order to improve the cost-effectiveness of the process, however, the research community has mainly focused on developing genome-wide sampling sequencing (GWSS) methods, a collection of reduced genome complexity sequencing, reduced genome representation sequencing and selective genome target sequencing. Here we review the major steps involved in library preparation, the types of adapters used for ligation and the primers designed for amplification of ligated products for sequencing. Unfortunately, currently available GWSS methods have their drawbacks, such as inconsistency in the number of reads per sample library, the number of sites/targets per individual, and the number of reads per site/target, all of which result in missing data. Suggestions are proposed here to improve library construction, genotype calling accuracy, genome-wide marker density and read mapping rate. In brief, optimized GWSS library preparation should generate a unique set of target sites with dense distribution along chromosomes and even coverage per site across all individuals.     

On the causes,consequences, and avoidance of PCR duplicates: Towards a theory of library complexity

Library preparation protocols for most sequencing technologies involve PCR amplification of the template DNA, which open the possibility that a given template DNA molecule is sequenced multiple times. Reads arising from this phenomenon, known as PCR duplicates, inflate the cost of sequencing and can jeopardize the reliability of affected experiments. Despite the pervasiveness of this artefact, our understanding of its causes and of its impact on downstream statistical analyses remains essentially empirical. Here, we develop a general quantitative model of amplification distortions in sequencing data sets, which we leverage to investigate the factors controlling the occurrence of PCR duplicates. We show that the PCR duplicate rate is determined primarily by the ratio between library complexity and sequencing depth, and that amplification noise (including in its dependence on the number of PCR cycles) only plays a secondary role for this artefact. We confirm our predictions using new and published RAD-seq libraries and provide a method to estimate library complexity and amplification noise in any data set containing PCR duplicates. We discuss how amplification-related artefacts impact downstream analyses, and in particular genotyping accuracy. The proposed framework unites the numerous observations made on PCR duplicates and will be useful to experimenters of all sequencing technologies where DNA availability is a concern.     

Single-cell genomic sequencing using Multiple Displacement Amplification

Single microbial cells can now be sequenced using DNA amplified by the Multiple Displacement Amplification (MDA) reaction. The few femtograms of DNA in a bacterium are amplified into micrograms of high molecular weight DNA suitable for DNA library construction and Sanger sequencing. The MDA-generated DNA also performs well when used directly as template for pyrosequencing by the 454 Life Sciences method. While MDA from single cells loses some of the genomic sequence, this approach will greatly accelerate the pace of sequencing from uncultured microbes. The genetically linked sequences from single cells are also a powerful tool to be used in guiding genomic assembly of shotgun sequences of multiple organisms from environmental DNA extracts (metagenomic sequences).     

Nanoliter reactors improve multiple displacement amplification of genomes from single cells

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-μl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.     

Sources of PCR-induced distortions in high-throughput sequencing data sets

PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules.     

Challenges with Using Primer IDs to Improve Accuracy of Next Generation Sequencing

Next generation sequencing technologies, like ultra-deep pyrosequencing (UDPS), allows detailed investigation of complex populations, like RNA viruses, but its utility is limited by errors introduced during sample preparation and sequencing. By tagging each individual cDNA molecule with barcodes, referred to as Primer IDs, before PCR and sequencing these errors could theoretically be removed. Here we evaluated the Primer ID methodology on 257,846 UDPS reads generated from a HIV-1 SG3Δenv plasmid clone and plasma samples from three HIV-infected patients. The Primer ID consisted of 11 randomized nucleotides, 4,194,304 combinations, in the primer for cDNA synthesis that introduced a unique sequence tag into each cDNA molecule. Consensus template sequences were constructed for reads with Primer IDs that were observed three or more times. Despite high numbers of input template molecules, the number of consensus template sequences was low. With 10,000 input molecules for the clone as few as 97 consensus template sequences were obtained due to highly skewed frequency of resampling. Furthermore, the number of sequenced templates was overestimated due to PCR errors in the Primer IDs. Finally, some consensus template sequences were erroneous due to hotspots for UDPS errors. The Primer ID methodology has the potential to provide highly accurate deep sequencing. However, it is important to be aware that there are remaining challenges with the methodology. In particular it is important to find ways to obtain a more even frequency of resampling of template molecules as well as to identify and remove artefactual consensus template sequences that have been generated by PCR errors in the Primer IDs.     

Octamer-primed sequencing technology: development of primer identification software

Octamer sequencing technology (OST) is a primer-directed sequencing strategy in which an individual octamer primer is selected from a pre-synthesized octamer primer library and used to sequence a DNA fragment. However, selecting candidate primers from such a library is time consuming and can be a bottleneck in the sequencing process. To accelerate the sequencing process and to obtain high quality sequencing data, a computer program, electronic OST or eOST, was developed to automatically identify candidate primers from an octamer primer library. eOST integrates the base calling software PHRED to provide a quality assessment for target sequences and identifies potential primer binding sites located within a high quality target region. To increase the sequencing success rate, eOST includes a simple dynamic folding algorithm to automatically calculate the free energy and predict the secondary structure within the template in the vicinity of the octamer-binding site. Several parameters were found to be important, including base quality threshold, the window size of the template sequence segment, and the threshold ΔG value. OST, coupled with the eOST software, can be used to sequence short DNA fragments or in the finishing assembly stage of large-scale sequencing of genomic DNA.     

Whole genome sequencing and its applications in medical genetics

Fundamental improvement was made for genome sequencing since the next-generation sequencing (NGS) came out in the 2000s. The newer technologies make use of the power of massively-parallel short-read DNA sequencing, genome alignment and assembly methods to digitally and rapidly search the genomes on a revolutionary scale, which enable large-scale whole genome sequencing (WGS) accessible and practical for researchers. Nowadays, whole genome sequencing is more and more prevalent in detecting the genetics of diseases, studying causative relations with cancers, making genome-level comparative analysis, reconstruction of human population history, and giving clinical implications and instructions. In this review, we first give a typical pipeline of whole genome sequencing, including the lab template preparation, sequencing, genome assembling and quality control, variants calling and annotations. We compare the difference between whole genome and whole exome sequencing (WES), and explore a wide range of applications of whole genome sequencing for both mendelian diseases and complex diseases in medical genetics. We highlight the impact of whole genome sequencing in cancer studies, regulatory variant analysis, predictive medicine and precision medicine, as well as discuss the challenges of the whole genome sequencing.      

Isothermal strand-displacement amplification applications for high-throughput genomics

Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using random hexamer primers with 29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.     

纳米孔测序信号处理及其在DNA数据存储的应用

随着高通量测序技术的不断更新,可以在单个分子水平读取核苷酸序列的第三代测序技术迅速发展,纳米孔测序技术是其具有代表性的单分子测序技术,该技术通过检测DNA单链分子穿过纳米孔时引起的跨膜电流信号的变化,实现碱基识别.纳米孔测序仪在便携性、碱基读取速度、测序读段长度等方面较传统的第一代与第二代测序技术都有明显优势.随着纳米...     

Imaging of DNA by scanning force microscopy.

The scanning probe microscopies applied to the sequencing of DNA is a challenging goal attempted by several groups. But one limitant parameter has been the sample preparation of DNA molecules. Here we report how to hold DNA molecules fixed on mica substrate and we show the three-dimensional configuration of double-stranded DNA obtained with our scanning force microscope. We can image DNA under negative supercoiling, a feature of general importance controlling the activities of DNA. We compared the electron micrographs of a carbon replica of the same DNA specimen with scanning force images which demonstrates well the feasibility and accuracy of our scanning probe measurements.     

Leveraging Identity-by-Descent for Accurate Genotype Inference in Family Sequencing Data

Sequencing family DNA samples provides an attractive alternative to population based designs to identify rare variants associated with human disease due to the enrichment of causal variants in pedigrees. Previous studies showed that genotype calling accuracy can be improved by modeling family relatedness compared to standard calling algorithms. Current family-based variant calling methods use sequencing data on single variants and ignore the identity-by-descent (IBD) sharing along the genome. In this study we describe a new computational framework to accurately estimate the IBD sharing from the sequencing data, and to utilize the inferred IBD among family members to jointly call genotypes in pedigrees. Through simulations and application to real data, we showed that IBD can be reliably estimated across the genome, even at very low coverage (e.g. 2X), and genotype accuracy can be dramatically improved. Moreover, the improvement is more pronounced for variants with low frequencies, especially at low to intermediate coverage (e.g. 10X to 20X), making our approach effective in studying rare variants in cost-effective whole genome sequencing in pedigrees. We hope that our tool is useful to the research community for identifying rare variants for human disease through family-based sequencing.     

Improved Multiple Displacement Amplification (iMDA) and Ultraclean Reagents

Background

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA).

Results

A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome.

Conclusions

The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.     

Optimization of asymmetric polymerase chain reaction for rapid fluorescent DNA sequencing

A high-throughput method for the preparation of single-stranded template DNA, which is suitable for sequence analysis using fluorescent labeling chemistry, is described here. In this procedure, the asymmetric polymerase chain reaction is employed to amplify recombinant plasmid or bacteriophage DNA directly from colonies or plaques. The use of amplification primers located at least 200 base pairs 5' to the site of sequencing primer annealing removes the need for extensive purification of the asymmetric polymerase chain reaction product. Instead, the single-stranded product DNA is purified by a simple isopropanol precipitation step and then directly sequenced using fluorescent dye-labeled oligonucleotides. This method significantly reduces the time and labor required for template preparation and improves fluorescent DNA sequencing strategies by providing a much more uniform yield of single-stranded DNA.     

Nonspecific PCR amplification by high-fidelity polymerases: implications for next-generation sequencing of AFLP markers

High-fidelity 'proofreading' polymerases are often used in library construction for next-generation sequencing projects, in an effort to minimize errors in the resulting sequence data. The increased template fidelity of these polymerases can come at the cost of reduced template specificity, and library preparation methods based on the AFLP technique may be particularly susceptible. Here, we compare AFLP profiles generated with standard Taq and two versions of a high-fidelity polymerase. We find that Taq produces fewer and brighter peaks than high-fidelity polymerase, suggesting that Taq performs better at selectively amplifying templates that exactly match the primer sequences. Because the higher accuracy of proofreading polymerases remains important for sequencing applications, we suggest that it may be more effective to use alternative library preparation methods.