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1.  Comparison of library preparation methods reveals their impact on interpretation of metatranscriptomic data 
BMC Genomics  2014;15(1):912.
Metatranscriptomics is rapidly expanding our knowledge of gene expression patterns and pathway dynamics in natural microbial communities. However, to cope with the challenges of environmental sampling, various rRNA removal and cDNA synthesis methods have been applied in published microbial metatranscriptomic studies, making comparisons arduous. Whereas efficiency and biases introduced by rRNA removal methods have been relatively well explored, the impact of cDNA synthesis and library preparation on transcript abundance remains poorly characterized. The evaluation of potential biases introduced at this step is challenging for metatranscriptomic samples, where data analyses are complex, for example because of the lack of reference genomes.
Herein, we tested four cDNA synthesis and Illumina library preparation protocols on a simplified mixture of total RNA extracted from four bacterial species. In parallel, RNA from each microbe was tested individually. cDNA synthesis was performed on rRNA depleted samples using the TruSeq Stranded Total RNA Library Preparation, the SMARTer Stranded RNA-Seq, or the Ovation RNA-Seq V2 System. A fourth experiment was made directly from total RNA using the Encore Complete Prokaryotic RNA-Seq. The obtained sequencing data were analyzed for: library complexity and reproducibility; rRNA removal efficiency and bias; the number of genes detected; coverage uniformity; and the impact of protocols on expression biases. Significant variations, especially in organism representation and gene expression patterns, were observed among the four methods. TruSeq generally performed best, but is limited by its requirement of hundreds of nanograms of total RNA. The SMARTer method appears the best solution for smaller amounts of input RNA. For very low amounts of RNA, the Ovation System provides the only option; however, the observed biases emphasized its limitations for quantitative analyses.
cDNA and library preparation methods may affect the outcome and interpretation of metatranscriptomic data. The most appropriate method should be chosen based on the available quantity of input RNA and the quantitative or non-quantitative objectives of the study. When low amounts of RNA are available, as in most metatranscriptomic studies, the SMARTer method seems to be the best compromise to obtain reliable results. This study emphasized the difficulty in comparing metatranscriptomic studies performed using different methods.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-912) contains supplementary material, which is available to authorized users.
PMCID: PMC4213505  PMID: 25331572
Metatranscriptomics; cDNA synthesis method; Gene expression
2.  Digital PCR provides sensitive and absolute calibration for high throughput sequencing 
BMC Genomics  2009;10:116.
Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing.
We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth.
The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.
PMCID: PMC2667538  PMID: 19298667
3.  Rapid DNA-Seq to Achieve High Coverage Libraries from 1ng – 1 μg in 2 Hours 
As next-generation sequencing technology progresses, the need for highly efficient, non-biased library generation for a wide variety of inputs has grown. High throughput sequencing operations require library preparation protocols which optimize many parameters, such as enzyme efficiency, low input flexibility and time efficiency without sacrificing the quality of downstream bioinformatics analysis. We've recently developed a highly efficient adapter ligation procedure that significantly increases the number of adapters bound to DNA insert. This should theoretically improve complexity, diversity, alignment and reduce the number of PCR duplicates in a library. Results: To test this, the enhanced adapter ligation step was used in our library prep protocol to prepare bacterial and human libraries along with currently established DNA-seq protocols. Final libraries were then analyzed on both Illumina MiSeq and HiSeq platforms, followed by coverage and library complexity analysis of the sequence data. Depth of coverage as a function of input material was shown to outperform regular ligation, especially in the 1 ng to 10 ng DNA input range. Normalized coverage as a function of GC content revealed no intrinsic bias when compared to older ligation and amplification methods. The enhanced adapter ligation and several new modifications in this new protocol allow users for the first time to build complete genomic DNA libraries in as little as 2 hours with low nanogram input material.
PMCID: PMC4162279
4.  Evaluation of Microarray Performance for Two RNA Amplification Methodologies 
Advances in high-throughput gene expression technologies such as microarrays have transformed our understanding of the molecular mechanisms underlying various types of biological processes and diseases. The Applause™ line of products (3'-Amp, WT-Amp ST, and WT-Amp Plus ST) addresses requirements of gene expression microarray users with high-quality RNA (50 nanograms) that want a low-cost, single-day, and reliable sample preparation solution. A comparison was made between the Applause line of amplification products and available vendor data for the GeneChip® WT cDNA Synthesis and Amplification and GeneChip3' IVT Express products. Data generated from 50 ng HeLa, MAQC A, and MAQC B RNA using the Applause 3'-Amp kit were compared to equivalent reported vendor data for the GeneChip 3' IVT Express amplification kit. NuGEN's Applause 3'-Amp outperformed the GeneChip 3' IVT Express with amplifications and labeling completed within 6 hours. It also did better than the GeneChip 3' IVT Express platform on HGU133 Plus 2.0 by percent Present Calls (%P). Differential expression analysis of MAQC samples demonstrated high correlation between the Applause 3'-Amp array data and data generated by quantitative PCR (qPCR) for the MAQC Project (Nature Biotech, 24:1151-1161 (2006)), with an R value of 0.96. Data generated from 50 ng brain and skeletal muscle RNA with the Applause WT-Amp ST and WT-Amp Plus ST kits were compared to equivalent 1 μg vendor data for GeneChip WT cDNA Synthesis and Amplification Kit. In addition to the improved speed (9 hours), the Applause WT-Amp kits also showed better QC array metrics (Pos-vs-Neg AUC, All-Probe-Set-Mean, All-Probe-Set-RLE-Mean) than the GeneChip WT cDNA Synthesis and Amplification kit. The Applause amplification system provides researchers with a fast, economical, and high-quality solution to everyday microarray needs.
PMCID: PMC2918120
5.  Multiplexed Illumina sequencing libraries from picogram quantities of DNA 
BMC Genomics  2013;14:466.
High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps.
We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment.
Application of this method allows whole genome studies from samples where material or yields are limiting.
PMCID: PMC3711846  PMID: 23837789
Illumina; ChIP-seq; Multiplex; Barcoding; Library preparation
6.  A comparison of RNA amplification techniques at sub-nanogram input concentration 
BMC Genomics  2009;10:326.
Gene expression profiling of small numbers of cells requires high-fidelity amplification of sub-nanogram amounts of RNA. Several methods for RNA amplification are available; however, there has been little consideration of the accuracy of these methods when working with very low-input quantities of RNA as is often required with rare clinical samples. Starting with 250 picograms-3.3 nanograms of total RNA, we compared two linear amplification methods 1) modified T7 and 2) Arcturus RiboAmp HS and a logarithmic amplification, 3) Balanced PCR. Microarray data from each amplification method were validated against quantitative real-time PCR (QPCR) for 37 genes.
For high intensity spots, mean Pearson correlations were quite acceptable for both total RNA and low-input quantities amplified with each of the 3 methods. Microarray filtering and data processing has an important effect on the correlation coefficient results generated by each method. Arrays derived from total RNA had higher Pearson's correlations than did arrays derived from amplified RNA when considering the entire unprocessed dataset, however, when considering a gene set of high signal intensity, the amplified arrays had superior correlation coefficients than did the total RNA arrays.
Gene expression arrays can be obtained with sub-nanogram input of total RNA. High intensity spots showed better correlation on array-array analysis than did unfiltered data, however, QPCR validated the accuracy of gene expression array profiling from low-input quantities of RNA with all 3 amplification techniques. RNA amplification and expression analysis at the sub-nanogram input level is both feasible and accurate if data processing is used to focus attention to high intensity genes for microarrays or if QPCR is used as a gold standard for validation.
PMCID: PMC2724417  PMID: 19619282
7.  Exome Sequencing from Nanogram Amounts of Starting DNA: Comparing Three Approaches 
PLoS ONE  2014;9(7):e101154.
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 SureSelectXT2 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.
PMCID: PMC4081514  PMID: 24992588
8.  Overview of the Agilent Technologies SureSelectTM Target Enrichment System 
Next-generation DNA sequencing has revolutionized the discovery of rare polymorphisms, structural variants, and novel transcripts. To meet the demand for fast, cost-effective, and accurate genome analysis methods from small scale studies to large sample cohorts, Agilent Technologies has developed the SureSelect™ Target Enrichment System. Available for the Illumina, SOLiD, and 454 NGS sequencing platforms, SureSelect is a highly robust, customizable, and scalable system that focuses analyses on specific genomic loci by in-solution hybrid capture. In addition, Agilent has introduced SureSelect XT for Illumina and SOLiD, which combines gDNA prep, library prep, and SureSelect Target Enrichment reagents in one complete kit. Both SureSelect and SureSelect XT demonstrate high performance, as measured by capture efficiency, uniformity, reproducibility, and SNP detection. We highlight the utility of the SureSelect system across a wide range of target sizes and genome complexity using pre-designed catalog libraries targeting cancer gene sets, sequences encoding the kinome, and both human and mouse All Exon content. In addition, user-defined custom content can be easily developed using the Agilent eArray software with candidate variant coordinates as input. User-defined content can be manufactured on-demand as a custom SureSelect kit, or combined with pre-defined Agilent catalog content using the Plus option. We propose a novel approach for variant discovery - using SureSelect catalog designs to uncover candidate variants, followed by the design of smaller focused custom libraries for SNP validation and region profiling. By pooling many samples together per lane or slide, SureSelect multiplexing kits for Illumina and SOLiD enable validation across large sample cohorts with substantial cost savings. Accurate post target enrichment pooling is facilitated by the Agilent Bioanalyzer and QPCR NGS Library Quantification kits which ensure equal representation across samples. Further efficiencies are realized using the Bravo Automated Liquid Handling Platform to meet the need for parallel preparation of multiplexed libraries.
PMCID: PMC3186662
9.  Enabling High-Throughput Discovery of the RNA Transcription Landscape Using a Directional RNA Workflow and a Combinatorial Multiplexing Approach 
Massively parallel next generation cDNA sequencing (RNA-Seq), has allowed many advances in the characterization and quantification of transcriptomes. In addition to enabling the detection of non-canonical transcription start sites and termination sites, alternative splice isoforms, transcript mutations and edits can be identified. Additionally, the ability to obtain information on the originating strand is useful for many reasons including for example: identification of antisense transcripts, determination of the transcribed strand of noncoding RNAs, and determination of expression levels of coding or noncoding overlapping transcripts. Overall, the ability to determine the originating strand can substantially enhance the value of a RNA-seq experiment. However, standard methods for sequencing RNA do not provide information on the DNA strand from which the RNA strand was transcribed, and methods for strand-specific library preparation can be inefficient and time-consuming. Our objective was to address this challenge by developing a streamlined, low input method for Directional RNA-Sequencing that highly retains strand orientation information while maintaining even coverage of transcript expression. This method is based on second strand labeling and excision after adaptor ligation; allowing differential tagging of the first strand cDNA ends. We have also extended the utility of this method by developing additional adaptor and primer reagents, including a dual barcoding approach that allows for multiplexing up to 96 samples. As a result, we have enabled highly multiplexed, strand-specific mRNA sequencing, as well as whole transcriptome sequencing (Total RNA-seq) from ribosomal-depleted samples, enabling the discovery of a much broader picture of expression dynamics including discovery of antisense transcripts. This work presents a streamlined, fast solution for complete RNA sequencing, with high quality data that illustrates the complexity and diversity of the RNA transcription landscape.
PMCID: PMC4162243
10.  DNA Library Preparation: Simultaneous DNA Fragmentation and Adaptor Tagging by In Vitro Transposition 
DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct DNA fragmentation, end-polishing, and adaptor-ligation steps; often with significant sample loss and hands-on time. EPICENTRE Biotechnologies has developed Nextera™ technology which combines these distinct steps into a single reaction; providing a streamlined, efficient, and high-throughput method for generating bar-coded sequencing libraries from nanogram amounts of DNA, in less than two hours. In vitro transposition with Nextera Transposomes simultaneously fragments and covalently tags the target DNA. Di-tagged sequencing libraries compatible with multiple sequencing platforms can be enriched and bar-coded using suppression PCR. Deep sequencing of Nextera libraries yields sequence depth, coverage, and accuracy that is comparable to control libraries produced using nebulization and standard protocols.
PMCID: PMC2918206
11.  Tapping diversity lost in transformations—in vitro amplification of ligation reactions 
Nucleic Acids Research  2006;34(16):e108.
Molecular evolution is a powerful means of engineering proteins. It usually requires the generation of a large recombinant DNA library of variants for cloning into a phage or plasmid vector, and the transformation of a host organism for expression and screening of the variant proteins. However, library size is often limited by the low yields of circular DNA and the poor transformation efficiencies of linear DNA. Here we have overcome this limitation by amplification of recombinant circular DNA molecules directly from ligation reactions. The amplification by bacteriophage Phi29 polymerase increased the number of transformants; thus from a nanogram-scale ligation of DNA fragments comprising two sub-libraries of variant antibody domains, we succeeded in amplifying a highly diverse and large combinatorial phage antibody library (>109 transformants in Escherichia coli and 105-fold more transformants than without amplification). From the amplified library, but not from the smaller un-amplified library, we could isolate several antibody fragments against a target antigen. It appears that amplification of ligations with Phi29 polymerase can help recover clones and molecular diversity otherwise lost in the transformation step. A further feature of the method is the option of using PCR-amplified vectors for ligations.
PMCID: PMC1636367  PMID: 16945952
12.  Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition 
Genome Biology  2010;11(12):R119.
We characterize and extend a highly efficient method for constructing shotgun fragment libraries in which transposase catalyzes in vitro DNA fragmentation and adaptor incorporation simultaneously. We apply this method to sequencing a human genome and find that coverage biases are comparable to those of conventional protocols. We also extend its capabilities by developing protocols for sub-nanogram library construction, exome capture from 50 ng of input DNA, PCR-free and colony PCR library construction, and 96-plex sample indexing.
PMCID: PMC3046479  PMID: 21143862
13.  Nucleic Acid Research Group 2013–2014 Study: Evaluating Library Synthesis Protocols for Sub-Nanogram ChIPSeq samples 
Chromatin immunoprecipitation followed by sequencing the precipitated DNA (ChIP-Seq) is the state-of-the-art method to study protein-DNA interactions. ChIP-Seq allows identification of binding sites of proteins across the entire genome in an unbiased manner. One of the major limitations of currently available ChIP-Seq protocols is the necessity to isolate sufficient amounts of immune precipitated DNA for subsequent sequencing. The NARG 2013/14 study evaluated library preparation alternatives starting from one and two orders of magnitude DNA less than standard protocols. Library preparation kits from seven different commercial providers were utilized in this project. Some of these kits were intended for low input while other kits were used outside of specifications of the manufacturers. Aliquots of the same preparation of ChIPed DNA were processed using the standard protocol for 10 ng of input DNA and the evaluated library preparation alternatives for 1 ng and 100 pg of input DNA. Each library type was prepared at two different ABRF Member labs and sequencing was done on a single Illumina HiSeq flow cell.
The results of low input compared to the standard protocol will be presented. The NARG 2013/14 study provides information how ChIP-Seq can be performed from 100 times less DNA than by standard methods with minimal compromising quality of results.
PMCID: PMC4162228
14.  Modification of the Transplex WTA2 Amplification Product for Next Generation Sequencing 
Transplex Whole Transcriptome Amplification (WTA2)a exponentially amplifies RNA producing a double-stranded cDNA library while precisely maintaining differential levels of individual transcripts in test and reference samples. Though originally designed to amplify nanogram quantities of RNA, Transplex WTA2 has been shown to be exceedingly effective for amplification from damaged RNA template (FFPE and laser captured tissue samples) and single-cell input quantities (picograms). The efficacy of Transplex WTA2 amplification for downstream applications, primarily qPCR and expression microarray analysis, is well-documented. It follows that the utilization of next-generation sequencing for gene expression research and diagnostics would be well served by Transplex amplification of RNA isolated from samples of severely restricted quantity or quality. Strategies for the integration of Transplex WTA2 with next-generation sequencing are examined, with particular emphasis on elimination of the characteristic fixed primer sequence associated with each amplicon in the amplification library. Removal of these sites will allow direct entry of the resulting product into the sequencing workflow. Methods under consideration will enable the WTA2 amplicon to feed into the current sample prep protocols for the Illumina GA and GAII, SoLiD 5500/5500xl, and Roche-454 GS FLX/Junior platforms.
PMCID: PMC3186653
15.  Evaluation of Exome Sequencing to Estimate Tumor Burden in Plasma 
PLoS ONE  2014;9(8):e104417.
Accurate estimation of systemic tumor load from the blood of cancer patients has enormous potential. One avenue is to measure the presence of cell-free circulating tumor DNA in plasma. Various approaches have been investigated, predominantly covering hotspot mutations or customized, patient-specific assays. Therefore, we investigated the utility of using exome sequencing to monitor circulating tumor DNA levels through the detection of single nucleotide variants in plasma. Two technologies, claiming to offer efficient library preparation from nanogram levels of DNA, were evaluated. This allowed us to estimate the proportion of starting molecules measurable by sequence capture (<5%). As cell-free DNA is highly fragmented, we designed and provide software for efficient identification of PCR duplicates in single-end libraries with a varying size distribution. On average, this improved sequence coverage by 38% in comparison to standard tools. By exploiting the redundant information in PCR-duplicates the background noise was reduced to ∼1/35000. By applying our optimized analysis pipeline to a simulation analysis, we determined the current sensitivity limit to ∼1/2400, starting with 30 ng of cell-free DNA. Subsequently, circulating tumor DNA levels were assessed in seven breast- and one prostate cancer patient. One patient carried detectable levels of circulating tumor DNA, as verified by break-point specific PCR. These results demonstrate exome sequencing on cell-free DNA to be a powerful tool for disease monitoring of metastatic cancers. To enable a broad implementation in the diagnostic settings, the efficiency limitations of sequence capture and the inherent noise levels of the Illumina sequencing technology must be further improved.
PMCID: PMC4136786  PMID: 25133800
16.  Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA 
Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina’s genome analyzer performs sequencing of a large number (> 107) of relatively short sequence reads (< 150 bp).The "paired end" approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined "T7LA" (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.
PMCID: PMC3227194  PMID: 22064688
17.  Improved Multiple Displacement Amplification (iMDA) and Ultraclean Reagents 
BMC Genomics  2014;15(1):443.
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).
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.
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.
PMCID: PMC4061449  PMID: 24906487
Whole genome amplification; Next generation sequencing; Multiple displacement amplification; Contamination; Clean reagents; DNA-free
18.  BA Fast Highly Multiplexed Solution to NGS Library Prep with Low Nanogram DNA Input 
As the quantity of data generated per next generation sequencing (NGS) run increases and the time required per run decreases, the ability to quickly produce and track large numbers of libraries is becoming increasingly important. In addition, the ability to produce high quality libraries from limited starting material and multiple sample types, including FFPE is essential. To overcome these challenges and to minimize the bottleneck of NGS library prep, we have developed a fast, streamlined DNA library preparation method using novel reagents and adaptors. This method accommodates a wide range of sample input quantities and types including genomic, ChIP and fragmented DNA (e.g. FFPE). Data analysis of libraries constructed from as little as 250 pg of ChIP DNA show high complexity and significant overlap of target peaks with libraries made from10 ng of DNA. We have extended the utility of this library prep method by developing additional adaptor and primer reagents. These include a dual barcoding approach that is compatible with Illumina library prep and our novel NEBNext adaptor. This approach enables multiplexing of up to 96 different samples, which can be used to increase the number of samples per flow cell, and/or to identify specific samples/libraries in a lab. Together, the simple, streamlined workflow and dual barcode approach, significantly reduces the turn-around time, enabling high throughput processing of samples for clinical analysis and large scale genomics studies.
PMCID: PMC4162240
19.  Improvement of PCR-free NGS Library Preparation to Obtain Uniform Read Coverage of Genome with Extremely High AT Content 
PCR amplification is commonly used in generating libraries for Next-Generation Sequencing (NGS) to efficiently enrich and amplify sequenceable DNA fragments. However, it introduces bias in the representation of the original complex template DNA. Such artifact has devastating effects in sequencing genomes with highly unbalanced base composition: regions of extremely high or low GC content, which are a substantial fraction of such genomes, are often covered with zero or near-zero read depth. PCR-free library preparation method has been published, which utilizes quantitative PCR and relies on previously sequenced similar libraries as standards to measure the amount of properly ligated fragments to obtain the library molecular concentration required by the sequencer. Here we present improvements of the PCR-free library preparation method to assess the efficiency of generating sequenceable library fragments and omit the quantitative PCR step. This is achieved by quantifying the ratio between properly ligated library fragments and improperly ligated fragments based on their fragment size difference and making use of the recently released Illumina TruSeq DNA Sample Prep kit that accommodates PCR-free library generation. Our improvement was applied to the highly AT-rich malaria parasite, Plasmodium falciparum, and the sequencing yield from Illumina HiSeq 2000 was optimal. Compared to the library generated with PCR amplification, our improvement increased the coverage uniformity across the whole P. falciparum genome of over 80% AT in a similar way as the published PCR-free method. Further enhancement is under our way to lower the input DNA amount to make the PCR-free library preparation method more widely applicable in genomic research.
PMCID: PMC3630566
20.  cDNA Library Generation for Transcriptome Analysis From Total RNA Equivalent to a Single Cell 
By providing sequence data on millions of short DNA fragments in parallel, Next Generation Sequencing has revolutionized biomedical research. In particular, the technique has enabled RNA expression analysis over the entire transcriptome with high sensitivity and dynamic range. With this has come a drive to utilize smaller sample inputs—with the goal of analyzing the transcriptome of a single cell.
One powerful method for cDNA preparation is SMART™ technology (Switching Mechanism At the 5' end of the RNA Template), which utilizes the template switching activity of reverse transcriptase to enable the direct addition of a PCR adaptor to the 3' end of the first-strand cDNA. The result is a single-tube protocol that enhances library amplification efficiency, while minimizing the chance for contamination, making it ideal for library preparation from small amounts of starting material. Indeed, the SMARTer™ Ultra Low RNA method allows researchers to readily obtain high quality data from as little as 10 pg of total RNA. Recent publications indicate that this technology is effective in single cell analysis.
Here we present data which demonstrates that SMART faithfully produces full-length cDNA for use as the template in library sample preparation. Sequencing results for libraries generated from 0.01 to 10 ng of mouse brain total RNA demonstrate that even with just 10 pg of input RNA, over 90% of the data maps to the genome, and the average transcript coverage is as uniform as that seen with much greater amounts of RNA. Also, under all conditions tested, rRNA reads accounted for only 3–5% of the total reads. Finally, comparing results to quantitative PCR for the MAQC (microarray quality control) gene set shows a high correlation with libraries made with 1 or 0.1 ng RNA. These data indicate that the SMART cDNA preparation method is an ideal choice for single cell transcriptome analysis.
PMCID: PMC3635253
21.  Coupling methanol denaturation, immobilized trypsin digestion, and accurate mass and time tagging for liquid chromatography-based shotgun proteomics of low nanogram amounts of RAW 264.7 cell lysate 
Analytical chemistry  2012;84(20):8715-8721.
We report the shotgun proteomic analysis of mammalian cell lysates that contain low nanogram amounts of protein. Proteins were denatured using methanol, digested using immobilized trypsin, and analyzed by UPLC-ESI-MS/MS. The approach generated more peptides and higher sequence coverage for a mixture of three standard proteins than the use of free trypsin solution digestion of heat- or urea-denatured proteins. We prepared triplicate RAW 264.7 cell lysates that contained 6 ng, 30 ng, 120 ng, and 300 ng of protein. An average of 2 ± 1, 23 ± 2, 134 ± 11, and 218 ± 26 proteins were detected for each sample size, respectively. The numbers of both protein and peptide IDs scaled linearly with the amount of sample taken for analysis. Our approach also outperformed traditional methods (free trypsin digestion of heat- or urea-denatured proteins) for 6 ng to 300 ng RAW 264.7 cell protein analysis in terms of number of peptides and proteins identified. The use of accurate mass and time (AMT) tags resulted in the identification of an additional 16 proteins based on 20 peptides from the 6 ng cell lysate prepared with our approach. When AMT analysis was performed for the 6 ng cell lysate prepared with traditional methods, no reasonable peptide signal could be obtained. In all cases, roughly ~30% of the digested sample was taken for analysis, corresponding to the analysis of a 2 ng aliquot of homogenate from the 6 ng cell lysate.
PMCID: PMC3477608  PMID: 22971241
22.  QPCR: a tool for analysis of mitochondrial and nuclear DNA damage in ecotoxicology 
Ecotoxicology (London, England)  2010;19(4):804-811.
The quantitative PCR (QPCR) assay for DNA damage and repair has been used extensively in laboratory species. More recently, it has been adapted to ecological settings. The purpose of this article is to provide a detailed methodological guide that will facilitate its adaptation to additional species, highlight its potential for ecotoxicological and biomonitoring work, and critically review the strengths and limitations of this assay. Major strengths of the assay include very low (nanogram to picogram) amounts of input DNA; direct comparison of damage and repair in the nuclear and mitochondrial genomes, and different parts of the nuclear genome; detection of a wide range of types of DNA damage; very good reproducibility and quantification; applicability to properly preserved frozen samples; simultaneous monitoring of relative mitochondrial genome copy number; and easy adaptation to most species. Potential limitations include the limit of detection (~1 lesion per 105 bases); the inability to distinguish different types of DNA damage; and the need to base quantification of damage on a control or reference sample. I suggest that the QPCR assay is particularly powerful for some ecotoxicological studies.
Electronic supplementary material
The online version of this article (doi:10.1007/s10646-009-0457-4) contains supplementary material, which is available to authorized users.
PMCID: PMC2844971  PMID: 20049526
Mitochondrial DNA; DNA damage; DNA repair; Genotoxicity; Biomarker
23.  Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes 
PLoS ONE  2011;6(3):e17625.
Technical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficultly in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics.
PMCID: PMC3062544  PMID: 21445340
24.  Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction 
BioTechniques  2013;54(1):25-34.
The amplification of DNA fragments, cloned between user-defined 5′ and 3′ end sequences, is a prerequisite step in the use of many current applications including massively parallel sequencing (MPS). Here we describe an improved method, called homopolymer tail-mediated ligation PCR (HTML-PCR), that requires very little starting template, minimal hands-on effort, is cost-effective, and is suited for use in high-throughput and robotic methodologies. HTML-PCR starts with the addition of homopolymer tails of controlled lengths to the 3′ termini of a double-stranded genomic template. The homopolymer tails enable the annealing-assisted ligation of a hybrid oligonucleotide to the template's recessed 5′ ends. The hybrid oligonucleotide has a user-defined sequence at its 5′ end. This primer, together with a second primer composed of a longer region complementary to the homopolymer tail and fused to a second 5′ user-defined sequence, are used in a PCR reaction to generate the final product. The user-defined sequences can be varied to enable compatibility with a wide variety of downstream applications. We demonstrate our new method by constructing MPS libraries starting from nanogram and sub-nanogram quantities of Vibrio cholerae and Streptococcus pneumoniae genomic DNA.
PMCID: PMC3605734  PMID: 23311318
molecular cloning; annealing-assisted ligation; DNA capture; massively-parallel sequencing
25.  Influence of Cultural Conditions on Mitomycin C-Mediated Bacteriophage Induction and Release of Salmonella Toxin 
Infection and Immunity  1981;32(1):232-242.
Several isolates of Salmonella were examined for the capacity to synthesize and release a cholera toxin-like toxin that exerted a biological effect on Chinese hamster ovary cells. Measurements of this Salmonella toxin, which was contained in cell sonic extracts and culture filtrates, were expressed in cholera toxin equivalents (nanograms), since the Chinese hamster ovary cell responses of the cholera toxin and the Salmonella toxin were indistinguishable. Comparative titrations of Salmonella preparations were also performed by using an enzyme-linked immunosorbent assay specific for cholera toxin antigen. The amount of Salmonella toxin synthesized was low (nanogram levels), but the toxin was detectable in cell sonic extracts as early as 6 h after culture inoculation and reached maximal levels by 12 h. Salmonella toxin antigen was not detectable in control culture filtrates until 48 h, but the addition of mitomycin C at 8.5 h resulted in the sudden appearance of toxin antigen at 10 to 12 h, and the toxin antigen level reached a maximum at 14 h. A large peak of Chinese hamster ovary cell activity was observed at 48 h in the control culture, but significant Chinese hamster ovary cell activity was detected as early as 14 h. A larger amount of Chinese hamster ovary cell-reactive material was observed as early as 10 h in cultures grown with mitomycin C. The mechanism of the mitomycin-mediated phenomenon that yielded more toxin in culture filtrates was associated with bacteriophage induction. A bacteriophage plaque assay with a susceptible Salmonella strain revealed that there were free bacteriophage in mitomycin-treated culture filtrates (but not control culture filtrates) at 12 h. Toxin production was greatest when cultures were grown at 30 to 37°C and lowest when cultures were grown at 25°C. The inoculum size and degree of culture aeration (agitation) had little effect on synthesis of the toxin, and toxin production occurred during anaerobic growth.
PMCID: PMC350612  PMID: 7012028

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