High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot

The potential importance of DNA methylation in the etiology of complex diseases has led to interest in the development of methylome-wide association studies (MWAS) aimed at interrogating all methylation sites in the human genome. When using blood as biomaterial for a MWAS the DNA is typically extracted directly from fresh or frozen whole blood that was collected via venous puncture. However, DNA extracted from dry blood spots may also be an alternative starting material. In the present study, we apply a methyl-CpG binding domain (MBD) protein enrichment-based technique in combination with next generation sequencing (MBD-seq) to assess the methylation status of the ~27 million CpGs in the human autosomal reference genome. We investigate eight methylomes using DNA from blood spots. This data are compared with 1,500 methylomes previously assayed with the same MBD-seq approach using DNA from whole blood. When investigating the sequence quality and the enrichment profile across biological features, we find that DNA extracted from blood spots gives comparable results with DNA extracted from whole blood. Only if the amount of starting material is ≤ 0.5µg DNA we observe a slight decrease in the assay performance. In conclusion, we show that high quality methylome-wide investigations using MBD-seq can be conducted in DNA extracted from archived dry blood spots without sacrificing quality and without bias in enrichment profile as long as the amount of starting material is sufficient. In general, the amount of DNA extracted from a single blood spot is sufficient for methylome-wide investigations with the MBD-seq approach.     

Methylome analysis using MeDIP-seq with low DNA concentrations

DNA methylation is an epigenetic mark that has a crucial role in many biological processes. To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input in order to minimize the drain on precious samples. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) fulfils these criteria, combining MeDIP with massively parallel DNA sequencing. Here we report an improved protocol using 100-fold less genomic DNA than that commonly used. We show comparable results for specificity (>97%) and enrichment (>100-fold) over a wide range of DNA concentrations (5,000-50 ng) and demonstrate the utility of the protocol for the generation of methylomes from rare bone marrow cells using 160-300 ng of starting DNA. The protocol described here, i.e., DNA extraction to generation of MeDIP-seq library, can be completed within 3-5 d.     

Semiconductor-based sequencing of genome-wide DNA methylation states

Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we developed a MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is low cost, rapid, and scalable. We applied this protocol to demonstrate MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450 BeadChip array, and accurately identifies sites of differential DNA methylation. Furthermore, we applied an integrative approach to further investigate and confirm the role of DNA methylation in alternative splicing and to profile 5mC and 5hmC variants of DNA methylation in normal human brain tissue that is localized over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions.     

Comparison of Methyl-capture Sequencing vs. Infinium 450K methylation array for methylome analysis in clinical samples

Interindividual variability in the epigenome has gained tremendous attention for its potential in pathophysiological investigation, disease diagnosis, and evaluation of clinical intervention. DNA methylation is the most studied epigenetic mark in epigenome-wide association studies (EWAS) as it can be detected from limited starting material. Infinium 450K methylation array is the most popular platform for high-throughput profiling of this mark in clinical samples, as it is cost-effective and requires small amounts of DNA. However, this method suffers from low genome coverage and errors introduced by probe cross-hybridization. Whole-genome bisulfite sequencing can overcome these limitations but elevates the costs tremendously. Methyl-Capture Sequencing (MC Seq) is an attractive intermediate solution to increase the methylome coverage in large sample sets. Here we first demonstrate that MC Seq can be employed using DNA amounts comparable to the amounts used for Infinium 450K. Second, to provide guidance when choosing between the 2 platforms for EWAS, we evaluate and compare MC Seq and Infinium 450K in terms of coverage, technical variation, and concordance of methylation calls in clinical samples. Last, since the focus in EWAS is to study interindividual variation, we demonstrate the utility of MC Seq in studying interindividual variation in subjects from different ethnicities.     

Exome Sequencing from Nanogram Amounts of Starting DNA: Comparing Three Approaches

Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelect^XT2 Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 µg of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input samples.     

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.     

Detection of differential DNA methylation in repetitive DNA of mice and humans perinatally exposed to bisphenol A

Developmental exposure to bisphenol A (BPA) has been shown to induce changes in DNA methylation in both mouse and human genic regions; however, the response in repetitive elements and transposons has not been explored. Here we present novel methodology to combine genomic DNA enrichment with RepeatMasker analysis on next-generation sequencing data to determine the effect of perinatal BPA exposure on repetitive DNA at the class, family, subfamily, and individual insertion level in both mouse and human samples. Mice were treated during gestation and lactation to BPA in chow at 0, 50, or 50,000 ng/g levels and total BPA was measured in stratified human fetal liver tissue samples as low (non-detect to 0.83 ng/g), medium (3.5 to 5.79 ng/g), or high (35.44 to 96.76 ng/g). Transposon methylation changes were evident in human classes, families, and subfamilies, with the medium group exhibiting hypomethylation compared to both high and low BPA groups. Mouse repeat classes, families, and subfamilies did not respond to BPA with significantly detectable differential DNA methylation. In human samples, 1251 individual transposon loci were detected as differentially methylated by BPA exposure, but only 19 were detected in mice. Of note, this approach recapitulated the discovery of a previously known mouse environmentally labile metastable epiallele, Cabp^IAP. Thus, by querying repetitive DNA in both mouse and humans, we report the first known transposons in humans that respond to perinatal BPA exposure.     

High resolution detection and analysis of CpG dinucleotides methylation using MBD-Seq technology

Methyl-CpG binding domain protein sequencing (MBD-seq) is widely used to survey DNA methylation patterns. However, the optimal experimental parameters for MBD-seq remain unclear and the data analysis remains challenging. In this study, we generated high depth MBD-seq data in MCF-7 cell and developed a bi-asymmetric-Laplace model (BALM) to perform data analysis. We found that optimal efficiency of MBD-seq experiments was achieved by sequencing ～100 million unique mapped tags from a combination of 500 mM and 1000 mM salt concentration elution in MCF-7 cells. Clonal bisulfite sequencing results showed that the methylation status of each CpG dinucleotides in the tested regions was accurately detected with high resolution using the proposed model. These results demonstrated the combination of MBD-seq and BALM could serve as a useful tool to investigate DNA methylome due to its low cost, high specificity, efficiency and resolution.     

Genomic sequencing by ligation-mediated PCR

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited due to the complexity of the mammalian genome. Ligation-mediated PCR (LMPCR) is a sensitive genomic sequencing procedure that generates high quality, reproducible sequence ladders starting with only 1 μg of uncloned mammalian DNA per reaction. This genomic sequencing procedure can be adapted for various methylation, in vivo footprinting and DNA adduct mapping procedures. We provide a detailed protocol for genomic sequencing by LMPCR and discuss the principles and applications of the method.     

Systematic assessment of reduced representation bisulfite sequencing to human blood samples: A promising method for large-sample-scale epigenomic studies

Complementary to the time- and cost-intensive direct bisulfite sequencing, we applied reduced representation bisulfite sequencing (RRBS) to the human peripheral blood mononuclear cells (PBMC) from YH, the Asian individual whose genome and epigenome has been deciphered in the YH project and systematically assessed the genomic coverage, coverage depth and reproducibility of this technology as well as the concordance of DNA methylation levels measured by RRBS and direct bisulfite sequencing for the detected CpG sites. Our result suggests that RRBS can cover more than half of CpG islands and promoter regions with a good coverage depth and the proportion of the CpG sites covered by the biological replicates reaches 80-90%, indicating good reproducibility. Given a smaller data quantity, RRBS enjoys much better coverage depth than direct bisulfite sequencing and the concordance of DNA methylation levels between the two methods is high. It can be concluded that RRBS is a time and cost-effective sequencing method for unbiased DNA methylation profiling of CpG islands and promoter regions in a genome-wide scale and it is the method of choice to assay certain genomic regions for multiple samples in a rapid way.     

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.     

The SSV-Seq 2.0 PCR-Free Method Improves the Sequencing of Adeno-Associated Viral Vector Genomes Containing GC-Rich Regions and Homopolymers

Adeno-associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co-transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high-throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV-Seq 2.0, a PCR-free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR-free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS-based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real-time PCR and are useful methods to improve the safety and efficacy of these viral vectors.     

GPMiner: an integrated system for mining combinatorial cis-regulatory elements in mammalian gene group

Background

Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences.

Results

We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates.

Conclusion

We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.     

Assessment of RainDrop BS-seq as a method for large-scale,targeted bisulfite sequencing

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.     

Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven’t benefited much from those next-generation sequencing technolo...     

Assessment of RainDrop BS-seq as a method for large-scale,targeted bisulfite sequencing

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.     

A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq

Background

Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template.

Results

The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5′ genomic termini and area immediately flanking the poly(C) region.

Conclusions

We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-828) contains supplementary material, which is available to authorized users.     

Assessing the utility of whole genome amplified DNA for next‐generation molecular ecology

DNA quantity can be a hindrance in ecological and evolutionary research programmes due to a range of factors including endangered status of target organisms, available tissue type, and the impact of field conditions on preservation methods. A potential solution to low‐quantity DNA lies in whole genome amplification (WGA) techniques that can substantially increase DNA yield. To date, few studies have rigorously examined sequence bias that might result from WGA and next‐generation sequencing of nonmodel taxa. To address this knowledge deficit, we use multiple displacement amplification (MDA) and double‐digest RAD sequencing on the grey mouse lemur (Microcebus murinus) to quantify bias in genome coverage and SNP calls when compared to raw genomic DNA (gDNA). We focus our efforts in providing baseline estimates of potential bias by following manufacturer's recommendations for starting DNA quantities (>100 ng). Our results are strongly suggestive that MDA enrichment does not introduce systematic bias to genome characterization. SNP calling between samples when genotyping both de‐novo and with a reference genome are highly congruent (>98%) when specifying a minimum threshold of 20X stack depth to call genotypes. Relative genome coverage is also similar between MDA and gDNA, and allelic dropout is not observed. SNP concordance varies based on coverage threshold, with 95% concordance reached at ~12X coverage genotyping de‐novo and ~7X coverage genotyping with the reference genome. These results suggest that MDA may be a suitable solution for next‐generation molecular ecological studies when DNA quantity would otherwise be a limiting factor.     

Q-RRBS: a quantitative reduced representation bisulfite sequencing method for single-cell methylome analyses

Reduced representation bisulfite sequencing (RRBS) is a powerful method of DNA methylome profiling that can be applied to single cells. However, no previous report has described how PCR-based duplication-induced artifacts affect the accuracy of this method when measuring DNA methylation levels. For quantifying the effects of duplication-induced artifacts on methylome profiling when using ultra-trace amounts of starting material, we developed a novel method, namely quantitative RRBS (Q-RRBS), in which PCR-induced duplication is excluded through the use of unique molecular identifiers (UMIs). By performing Q-RRBS on varying amounts of starting material, we determined that duplication-induced artifacts were more severe when small quantities of the starting material were used. However, through using the UMIs, we successfully eliminated these artifacts. In addition, Q-RRBS could accurately detect allele-specific methylation in absence of allele-specific genetic variants. Our results demonstrate that Q-RRBS is an optimal strategy for DNA methylation profiling of single cells or samples containing ultra-trace amounts of cells.