Synthesis cost-optimal targeted mutant protein libraries.

Protein variant libraries produced by site-directed mutagenesis are a useful tool utilized by protein engineers to explore variants with potentially improved properties, such as activity and stability. These libraries are commonly built by selecting residue positions and alternative beneficial mutations for each position. All possible combinations are then constructed and screened, by incorporating degenerate codons at mutation sites. These degenerate codons often encode additional unwanted amino acids or even STOP codons. Our study aims to take advantage of annealing based recombination of oligonucleotides during synthesis and utilize multiple degenerate codons per mutation site to produce targeted protein libraries devoid of unwanted variants. Toward this goal we created an algorithm to calculate the minimum number of degenerate codons necessary to specify any given amino acid set, and a dynamic programming method that uses this algorithm to optimally partition a DNA target sequence with degeneracies into overlapping oligonucleotides, such that the total cost of synthesis of the target mutant protein library is minimized. Computational experiments show that, for a modest increase in DNA synthesis costs, beneficial variant yields in produced mutant libraries are increased by orders of magnitude, an effect particularly pronounced in large combinatorial libraries.

Highly Parallelized Construction of DNA from Low-Cost Oligonucleotide Mixtures Using Data-Optimized Assembly Design and Golden Gate.

Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically synthesized microarray-derived oligonucleotides. Here, we apply Data-optimized Assembly Design (DAD) to the synthesis of hundreds of codon-optimized genes in both constitutive and inducible vectors using Golden Gate Assembly. Starting from oligonucleotide pools, we synthesize genes in three simple steps: (1) amplification of parts belonging to individual assemblies in parallel from a single pool; (2) Golden Gate Assembly of parts for each construct; and (3) transformation. We construct genes from receiving DNA to sequence confirmed isolates in as little as 4 days. By leveraging the ligation fidelity afforded by T4 DNA ligase, we expect to be able to construct a larger breadth of sequences not currently supported by homology-based methods, which require stability of extensive single-stranded DNA overhangs.

Cost-Efficient Sequence-Based Nonextensible Oligonucleotide in Real-Time PCR and High-Throughput Sequencing.

Molecular detection of disease-associated mutations, especially those with low abundance, is essential for academic research and clinical diagnosis. Certain variant detection methods reach satisfactory sensitivity and specificity in detecting rare mutations based on the introduction of blocking oligos to prevent the amplification of wild-type or unwanted templates, thus selectively amplifying and enriching the mutations. These blocking oligos usually suppress PCR amplification through the 3' chemical modifications, with high price, slow synthesis, and reduced purity. Herein, we introduce chemistry-free designs to block enzymatic extension during PCR by the steric hindrance from the secondary structures attached to the 3' end of the oligos (nonextensible oligonucleotide, NEO). We demonstrated that NEO efficiently prohibited the extension of both Taq and high-fidelity DNA polymerases. By further applying NEO as blockers in blocker displacement amplification (BDA) qPCR, multiplex BDA (mBDA) NGS, and quantitative BDA (QBDA) NGS methods, we showed that NEO blockers had performance comparable with previously validated chemical modifications. Comparison experiments using QBDA with NEO blockers and droplet digital PCR (ddPCR) on clinical formalin-fixed paraffin-embedded (FFPE) samples exhibited 100% concordance. Lastly, the ability of NEO to adjust plex uniformity through changes of PCR amplification efficiency was demonstrated in an 80-plex NGS panel.

Outcomes of the European Federation of Pharmaceutical Industries and Associations Oligonucleotide Working Group Survey on Nonclinical Practices and Regulatory Expectations for Therapeutic Oligonucleotide Safety Assessment.

The Oligonucleotide Working Group of the European Federation of Pharmaceutical Industries and Associations (EFPIA) conducted a survey of companies to understand the trends in nonclinical practices and regulatory expectations for oligonucleotide drug safety assessment. Twenty-two companies of different types, with varying oligonucleotide experience levels in the field, participated. The survey identified key regulatory challenges and areas of perceived health authority (HA) concern regarding nonclinical safety strategies for oligonucleotides, such as the choice of toxicology species, approaches to dose setting in toxicity studies, dose scaling from animals to humans, the implementation (and regulatory acceptability) of lean packages, and methods for dealing with impurities and human-specific off-targets. The perceived oligonucleotide experience of HAs and the relevance of guidance to oligonucleotide development were also assessed. The results showed a general lack of consensus on nonclinical safety assessment approaches being used for this growing class of medicines and highlight the need for continuing collaboration between sponsors and HAs to better define best practices.

Oligonucleotides to the (Gene) Rescue: FDA Approvals 2017-2019.

Four decades have passed since oligonucleotides were first used to manipulate gene expression. There were few FDA approvals prior to 2016, mostly of drugs that eventually exhibited poor performance in the market. The aura of their younger siRNA relatives had also faded during the past 15 years. However, several FDA approvals have occurred in the past 4 years, restoring hope that a new era has dawned in oligonucleotide/siRNA clinical therapeutics. Here, we review the field of oligonucleotide therapeutics and provide an update on FDA approvals of oligonucleotides from 2017 until the second quarter of 2019. We take into consideration the ethical issues looming over the still somewhat limited efficacy of these molecules, the toxicity of treatment, and the exorbitant cost of these therapeutic agents, which limits accessibility for many.

Quantitative assessment of LASSO probe assembly and long-read multiplexed cloning.

BACKGROUND: Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences. LASSO dramatically improves the capture length limit of current DNA padlock probe technology from approximately 150 bps to several kbps. High-throughput LASSO capture involves the parallel assembly of thousands of probes. However, malformed probes are indiscernible from properly formed probes using gel electrophoretic techniques. Therefore, we used next-generation sequencing (NGS) to assess the efficiency of LASSO probe assembly and how it relates to the nature of DNA capture and amplification. Additionally, we introduce a simplified single target LASSO protocol using classic molecular biology techniques for qualitative and quantitative assessment of probe specificity. RESULTS: A LASSO probe library targeting 3164 unique E. coli ORFs was assembled using two different probe assembly reaction conditions with a 40-fold difference in DNA concentration. Unique probe sequences are located within the first 50 bps of the 5' and 3' ends, therefore we used paired-end NGS to assess probe library quality. Properly mapped read pairs, representing correctly formed probes, accounted for 10.81 and 0.65% of total reads, corresponding to ~ 80% and ~ 20% coverage of the total probe library for the lower and higher DNA concentration conditions, respectively. Subsequently, we used single-end NGS to correlate probe assembly efficiency and capture quality. Significant enrichment of LASSO targets over non-targets was only observed for captures done using probes assembled with a lower DNA concentration. Additionally, semi-quantitative polyacrylamide gel electrophoresis revealed a ~ 10-fold signal-to-noise ratio of LASSO capture in a simplified system. CONCLUSIONS: These results suggest that LASSO probe coverage for target sequences is more predictive of successful capture than probe assembly depth-enrichment. Concomitantly, these results demonstrate that DNA concentration at a critical step in the probe assembly reaction significantly impacts probe formation. Additionally, we show that a simplified LASSO capture protocol coupled to PAGE (polyacrylamide gel electrophoresis) is highly specific and more amenable to small-scale LASSO approaches, such as screening novel probes and templates.

An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA.

As the applications of CRISPR-Cas9 technology diversify and spread beyond the laboratory to diagnostic and therapeutic use, the demands of gRNA synthesis have increased and access to tailored gRNAs is now restrictive. Enzymatic routes are time-consuming, difficult to scale-up and suffer from polymerase-bias while existing chemical routes are inefficient. Here, we describe a split-and-click convergent chemical route to individual or pools of sgRNAs. The synthetic burden is reduced by splitting the sgRNA into a variable DNA/genome-targeting 20-mer, produced on-demand and in high purity, and a fixed Cas9-binding chemically-modified 79-mer, produced cost-effectively on large-scale, a strategy that provides access to site-specific modifications that enhance sgRNA activity and in vivo stability. Click ligation of the two components generates an artificial triazole linkage that is tolerated in functionally critical regions of the sgRNA and allows efficient DNA cleavage in vitro as well as gene-editing in cells with no unexpected off-target effects.

High-throughput thermal stability assessment of DNA hairpins based on high resolution melting.

On the basis of high-resolution melting, a high-throughput approach to measure melting temperatures (Tms) of short DNA hairpins was developed. With this method, Tms of thousands of triloop, tetraloop, and pentaloop hairpins involving various loop sequences and various closing base pairs (cbp) were obtained in hours. The stability of triloop hairpins decreased with the change of cbp (5'-3') in the order of c-g > g-c > t-a ≥ a-t, showing that the cbp of 5'-Pyr-Pur-3' (Pyr = pyrimidine, Pur = purine) contributed more stability than 5'-Pur-Pyr-3'. For tetraloop hairpins, GNNA, GNAB, and CNNG (N = A, G, C, or T; B = G, C, or T) were found to be highly stable irrespective of the cbp type. TNNA was also stable in both g-c and a-t families, while CGNA only in the c-g family. Pentaloop hairpins of cTGNAGg, cGNYNAg (Y = T or C) and cCGNNAg were exceptionally stable motifs. In most cases, pyrimidine-rich loops were more favorable to stabilize the whole structure than purine-rich ones. The present approach showed a good performance in assessing the thermal stability of large amounts of DNA hairpins comprehensively. These data are useful to understand the sequence dependence of the stability of DNA secondary structures and promising to improve the structure simulation by consummating basic databases.

Wild­type blocking pcr coupled with internal competitive amplified fragment improved the detection of rare mutation of KRAS.

Mutant KRAS proto­oncogene GTPase (KRAS) serves an important role in predicting the development, diagnosis, treatment and efficacy of targeted drug therapies for colorectal cancer. To improve the detection efficacy of trace amount of mutant KRAS, the locked nucleic acid­based method was modified in the present study. Internal competitive amplification fragments were used to improve the inhibition of wild­type KRAS with a wild­type blocking (WTB) probe and specifically amplify the trace amounts of mutant KRAS. The modified method, quantitative clamp­based polymerase chain reaction technology using WTB coupled with internal competitive reference to enhance the amplification specificity, named WIRE­PCR, completely blocked the amplification of wild­type KRAS in 50­150 ng DNA templates. The added internal competitive amplified fragments were amplified together with the target gene, which were used to reduce base mismatch due to the high number of cycles in PCR and quantify the total amount of DNA. The results demonstrated that WIRE­PCR facilitated the detection of mutated alleles at a single molecular level. In the colorectal biopsies from 50 patients with suspected colorectal cancer, 18 cases (36%) contained mutant KRAS, and the amount of mutant DNA accounted for 18.6­64.2% of the total DNA. WIRE­PCR is a simple, rapid and low­cost quantitative analysis method for the detection of trace amounts of the mutant KRAS.

Interactive analysis and assessment of single-cell copy-number variations.

We present Ginkgo (http://qb.cshl.edu/ginkgo), a user-friendly, open-source web platform for the analysis of single-cell copy-number variations (CNVs). Ginkgo automatically constructs copy-number profiles of cells from mapped reads and constructs phylogenetic trees of related cells. We validated Ginkgo by reproducing the results of five major studies. After comparing three commonly used single-cell amplification techniques, we concluded that degenerate oligonucleotide-primed PCR is the most consistent for CNV analysis.

Sudden Cardiac Death in Brazil: A Community-Based Autopsy Series (2006-2010) / Morte Súbita Cardíaca no Brasil: Análise dos Casos de Ribeirão Preto (2006-2010)

Background: Sudden cardiac death (SCD) is a sudden unexpected event, from a cardiac cause, that occurs in less than one hour after the symptoms onset, in a person without any previous condition that would seem fatal or who was seen without any symptoms 24 hours before found dead. Although it is a relatively frequent event, there are only few reliable data in underdeveloped countries. Objective: We aimed to describe the features of SCD in Ribeirão Preto, Brazil (600,000 residents) according to Coroners’ Office autopsy reports. Methods: We retrospectively reviewed 4501 autopsy reports between 2006 and 2010, to identify cases of SCD. Specific cause of death as well as demographic information, date, location and time of the event, comorbidities and whether cardiopulmonary resuscitation (CPR) was attempted were collected. Results: We identified 899 cases of SCD (20%); the rate was 30/100000 residents per year. The vast majority of cases of SCD involved a coronary artery disease (CAD) (64%) and occurred in men (67%), between the 6th and the 7th decades of life. Most events occurred during the morning in the home setting (53.3%) and CPR was attempted in almost half of victims (49.7%). The most prevalent comorbidity was systemic hypertension (57.3%). Chagas’ disease was present in 49 cases (5.5%). Conclusion: The majority of victims of SCD were men, in their sixties and seventies and the main cause of death was CAD. Chagas’ disease, an important public health problem in Latin America, was found in about 5.5% of the cases. .

Fundamento: Morte súbita cardíaca (MSC) é um evento súbito e inesperado, de causa cardiovascular, que ocorre em menos de uma hora após o início dos sintomas, em indivíduo sem qualquer condição clínica prévia potencialmente fatal ou assintomático nas últimas 24 horas antes do óbito, em caso de morte não testemunhada. Apesar de ser um evento relativamente frequente, há poucos dados confiáveis na literatura sobre países em desenvolvimento. Objetivo: Descrever as características da MSC em Ribeirão Preto (SP 600.000 habitantes) baseando-se nos relatórios de autopsias do Serviço de Verificação de Óbitos do Interior. Métodos: Foram revisados retrospectivamente 4.501 relatórios de autopsias entre 2006 e 2010, para identificar casos de MSC. Foram coletados dados como causa específica do óbito, características demográficas e comorbidades das vítimas, data, local e hora do evento, e se foram realizadas manobras de ressuscitação cardiopulmonar (RCP). Resultados: Foram identificados 899 casos de MSC (20%; razão 30/100.000 habitantes por ano). A principal causa de MSC foi doença arterial coronariana (DAC - 64%), acometendo homens (67%) entre a sexta e a sétima década de vida. A maior parte dos eventos ocorreu durante a manhã, no domicílio (53,3%), e a RCP foi realizada em quase metade das vítimas (49,7%). A comorbidade mais prevalente foi hipertensão arterial sistêmica (57,3%). Doença de Chagas foi detectada em 49 casos (5,5%). Conclusão: A maioria dos casos de MSC ocorreu por DAC em homens entre a sexta e a sétima década de vida. Doença de Chagas, um importante problema de saúde pública na América Latina, foi detectada em 5,5% dos casos. .

Chromogen detection of microRNA in frozen clinical tissue samples using LNA™ probe technology.

Specific chromogen- and fluorescence-based detection of microRNA by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded (FFPE) tissue sections has been facilitated by locked nucleic acid (LNA)-based probe technology and can be performed within a single working day. In the current method paper, we present a similar simple 1-day ISH method developed for cryostat sections obtained from clinical cryo-embedded tissue samples. The presented chromogen-based ISH method does not involve proteolytic pretreatment, which is mandatory for FFPE sections, but still retains a sensitivity level similar to that obtained in FFPE sections. The LNA-based ISH method is not only applicable in situations where only access to cryo-embedded material is possible, but it also has a potential use if combining microRNA ISH with immunohistochemistry in double fluorescence staining with antibodies not being compatible with proteolytic predigestion.

Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

Development of an oligonucleotide-based fluorescence assay for the identification of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors.

Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3'-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.

Molecular assessment, drug-resistant profile, and spacer oligonucleotide typing (spoligotyping) of Mycobacterium tuberculosis strains from Tamaulipas, México.

BACKGROUND: Tuberculosis remains a serious global health problem involving one-third of the world population. A wide diversity of Mycobacterium tuberculosis strains cause about 1.5 million deaths/year worldwide, but in developing countries, the genetic diversity of M. tuberculosis strains remains largely unknown. We conducted a first insight into the population diversity of M. tuberculosis strains from Tamaulipas, Mexico. METHODS: Seventy-two M. tuberculosis strains were identified and genetic diversity determined by spoligotyping. Drug sensibility testing and punctual mutations in inhA, ahpC, rpoB, and katG genes were assessed. RESULTS: Spoligotyping analysis showed a higher prevalence of LAM9 > T1 > Haarlem3 subfamilies among 53 spoligotype patterns. Unexpectedly, five Beijing strains conforming four unique spoligopatterns were recovered. The more frequently isolated strains (LAM9 and T1), but none of the Beijing strains, were found resistant to INH or RIF. Also, no drug resistance was found among Haarlem3 isolates. The katG(315) gene mutation was found in 83% of INH-resistant strains, whereas rpoB(526) were associated in only 43% of RIF M. tuberculosis drug-resistant strains. CONCLUSIONS: This and other studies report a high rate of orphan spoligotypes, which highlights the need for genotyping implementation as a routine technique for better understanding of M. tuberculosis strains in developing countries such as Mexico.

Genotyping by genome reducing and sequencing for outbred animals.

Next-generation sequencing (NGS) approaches are widely used in genome-wide genetic marker discovery and genotyping. However, current NGS approaches are not easy to apply to general outbred populations (human and some major farm animals) for SNP identification because of the high level of heterogeneity and phase ambiguity in the haplotype. Here, we reported a new method for SNP genotyping, called genotyping by genome reducing and sequencing (GGRS) to genotype outbred species. Through an improved procedure for library preparation and a marker discovery and genotyping pipeline, the GGRS approach can genotype outbred species cost-effectively and high-reproducibly. We also evaluated the efficiency and accuracy of our approach for high-density SNP discovery and genotyping in a large genome pig species (2.8 Gb), for which more than 70,000 single nucleotide polymorphisms (SNPs) can be identified for an expenditure of only $80 (USD)/sample.

Genotyping-by-sequencing (GBS): a novel, efficient and cost-effective genotyping method for cattle using next-generation sequencing.

High-throughput genotyping methods have increased the analytical power to study complex traits but high cost has remained a barrier for large scale use in animal improvement. We have adapted genotyping-by-sequencing (GBS) used in plants for genotyping 47 animals representing 7 taurine and indicine breeds of cattle from the US and Africa. Genomic DNA was digested with different enzymes, ligated to adapters containing one of 48 unique bar codes and sequenced by the Illumina HiSeq 2000. PstI was the best enzyme producing 1.4 million unique reads per animal and initially identifying a total of 63,697 SNPs. After removal of SNPs with call rates of less than 70%, 51,414 SNPs were detected throughout all autosomes with an average distance of 48.1 kb, and 1,143 SNPs on the X chromosome at an average distance of 130.3 kb, as well as 191 on unmapped contigs. If we consider only the SNPs with call rates of 90% and over, we identified 39,751 on autosomes, 850 on the X chromosome and 124 on unmapped contigs. Of these SNPs, 28,843 were not tightly linked to other SNPs. Average marker density per autosome was highly correlated with chromosome size (coefficient of correlation = -0.798, r(2) = 0.637) with higher density in smaller chromosomes. Average SNP call rate was 86.5% for all loci, with 53.0% of the loci having call rates >90% and the average minor allele frequency being 0.212. Average observed heterozygosity ranged from 0.046-0.294 among individuals, and from 0.064-0.197 among breeds, with Brangus showing the highest diversity as expected. GBS technique is novel, flexible, sufficiently high-throughput, and capable of providing acceptable marker density for genomic selection or genome-wide association studies at roughly one third of the cost of currently available genotyping technologies.

Pre-capture multiplexing improves efficiency and cost-effectiveness of targeted genomic enrichment.

BACKGROUND: Targeted genomic enrichment (TGE) is a widely used method for isolating and enriching specific genomic regions prior to massively parallel sequencing. To make effective use of sequencer output, barcoding and sample pooling (multiplexing) after TGE and prior to sequencing (post-capture multiplexing) has become routine. While previous reports have indicated that multiplexing prior to capture (pre-capture multiplexing) is feasible, no thorough examination of the effect of this method has been completed on a large number of samples. Here we compare standard post-capture TGE to two levels of pre-capture multiplexing: 12 or 16 samples per pool. We evaluated these methods using standard TGE metrics and determined the ability to identify several classes of genetic mutations in three sets of 96 samples, including 48 controls. Our overall goal was to maximize cost reduction and minimize experimental time while maintaining a high percentage of reads on target and a high depth of coverage at thresholds required for variant detection. RESULTS: We adapted the standard post-capture TGE method for pre-capture TGE with several protocol modifications, including redesign of blocking oligonucleotides and optimization of enzymatic and amplification steps. Pre-capture multiplexing reduced costs for TGE by at least 38% and significantly reduced hands-on time during the TGE protocol. We found that pre-capture multiplexing reduced capture efficiency by 23 or 31% for pre-capture pools of 12 and 16, respectively. However efficiency losses at this step can be compensated by reducing the number of simultaneously sequenced samples. Pre-capture multiplexing and post-capture TGE performed similarly with respect to variant detection of positive control mutations. In addition, we detected no instances of sample switching due to aberrant barcode identification. CONCLUSIONS: Pre-capture multiplexing improves efficiency of TGE experiments with respect to hands-on time and reagent use compared to standard post-capture TGE. A decrease in capture efficiency is observed when using pre-capture multiplexing; however, it does not negatively impact variant detection and can be accommodated by the experimental design.

Molecular techniques in ecohealth research toolkit: facilitating estimation of aggregate gastroenteritis burden in an irrigated periurban landscape.

Assessment of microbial hazards associated with certain environmental matrices, livelihood strategies, and food handling practices are constrained by time-consuming conventional microbiological techniques that lead to health risk assessments of narrow geographic or time scope, often targeting very few pathogens. Health risk assessment based on one or few indicator organisms underestimates true disease burden due a number of coexisting causative pathogens. Here, we employed molecular techniques in a survey of Cryptosporidium parvum, Giardia lamblia, Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., Shigella spp., Vibrio cholera, and Rotavirus A densities in canal water with respect to seasonality and spatial distribution of point-nonpoint pollution sources. Three irrigational canals stretching across nearly a 150-km(2) periurban landscape, traditionally used for agricultural irrigation but function as vital part of municipal wastewater stabilization in recent years, were investigated. Compiled stochastic data (pathogen concentration, susceptible populations) and literature-obtained deterministic data (pathogen dose-response model parameter values) were used in estimating waterborne gastroenteritis burden. Exposure scenarios include swimming or fishing, consuming canal water-irrigated vegetables, and ingesting or inhaling water aerosols while working in canal water-irrigated fields. Estimated annual gastroenteritis burden due individual pathogens among the sampling points was -10.6log(10) to -2.2log(10) DALYs. Aggregated annual gastroenteritis burden due all the target pathogens per sampling point was -3.1log(10) to -1.9log(10) DALYs, far exceeding WHO acceptable limit of -6.0log(10) DALYs. The present approach will facilitate the comprehensive collection of surface water microbiological baseline data and setting of benchmarks for interventions aimed at reducing microbial hazards in similar landscapes worldwide.

High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment.

The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.