Efficient depletion of host DNA contamination in malaria clinical sequencing.

The cost of whole-genome sequencing (WGS) is decreasing rapidly as next-generation sequencing technology continues to advance, and the prospect of making WGS available for public health applications is becoming a reality. So far, a number of studies have demonstrated the use of WGS as an epidemiological tool for typing and controlling outbreaks of microbial pathogens. Success of these applications is hugely dependent on efficient generation of clean genetic material that is free from host DNA contamination for rapid preparation of sequencing libraries. The presence of large amounts of host DNA severely affects the efficiency of characterizing pathogens using WGS and is therefore a serious impediment to clinical and epidemiological sequencing for health care and public health applications. We have developed a simple enzymatic treatment method that takes advantage of the methylation of human DNA to selectively deplete host contamination from clinical samples prior to sequencing. Using malaria clinical samples with over 80% human host DNA contamination, we show that the enzymatic treatment enriches Plasmodium falciparum DNA up to ∼9-fold and generates high-quality, nonbiased sequence reads covering >98% of 86,158 catalogued typeable single-nucleotide polymorphism loci

Sources of DNA contamination and decontamination procedures in the forensic laboratory

The sensitivity of forensic DNA typing techniques can cause problems when evidence samples are inadvertently contaminated with DNA from another source. Therefore, precautions need to be taken to minimize the risk of contamination. In this study, laboratory air and surfaces, tools and equipment were evaluated as potential sources of contaminating DNA. Subsequently, two decontamination procedures, i.e. the conventionally used sodium hypochlorite and the commercially available DNA decontamination solution DNA ZAPTM (Applied Biosystems), were compared for their use in removing potentially contaminating DNA from the laboratory working environment. From our results, it can be concluded that air is unlikely to be the source of observed DNA contamination in the laboratory whereas DNA accumulating on surfaces, tools and equipment within the laboratory environment may potentially be transferred to evidence samples. DNA ZAPTM outperformed the conventionally used sodium hypochlorite decontamination procedure. Stringent preventive measures and decontamination of equipment and laboratory surfaces is important to avoid secondary transfer of this contaminating DNA to evidence samples

Reducing Contamination in Forensic Science

The sensitivity of modern forensic techniques has drastically increased, with sensitive technology detecting even the smallest traces of DNA evidence left behind. This has made it possible to detect DNA profiles deposited through contamination. When DNA contamination occurs in forensic science, it has the potential to change the outcome of a criminal investigation and may have significant social and financial repercussions. A compilation of global research shows that DNA evidence transfer can occur during forensic product manufacturing, the fingerprinting process, or even autopsy and crime lab examinations. These vital areas of the forensic investigation are vulnerable to contamination, and national standards should address this susceptibility. Understanding the origins of contamination events provides the greatest insight into preventing their occurrence and maintaining the integrity of forensic evidence

An optimized microarray platform for assaying genomic variation in Plasmodium falciparum field populations

We present an optimized probe design for copy number variation (CNV) and SNP genotyping in the Plasmodium falciparum genome. We demonstrate that variable length and isothermal probes are superior to static length probes. We show that sample preparation and hybridization conditions mitigate the effects of host DNA contamination in field samples. The microarray and workflow presented can be used to identify CNVs and SNPs with 95% accuracy in a single hybridization, in field samples containing up to 92% human DNA contamination

Present-Day DNA contamination in ancient DNA datasets

Abstract Present-day contamination can lead to false conclusions in ancient DNA studies. A number of methods are available to estimate contamination, which use a variety of signals and are appropriate for different types of data. Here an overview of currently available methods highlighting their strengths and weaknesses is provided, and a classification based on the signals used to estimate contamination is proposed. This overview aims at enabling researchers to choose the most appropriate methods for their dataset. Based on this classification, potential avenues for the further development of methods are discussed

Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses

Extraction of high quality RNA is a prerequisite for downstream application in functional genomics analyses. However, the extraction and purification of pure nucleic acids from root tissues is generally difficult due to the high concentration of carbohydrates and secondary metabolites. Furthermore, the presence of enzymatic inhibitors such as fulvic and humic acids can also negatively affect extraction quality, when extracting from clay soil-grown roots. In this work, total RNA was extracted from soil-grown roots of Ilex paraguariensis using four commercially available kits: SpectrumTM, RNeasy®, TRI Reagent®, and SV Total RNA Isolation System. Spectral measurement and electrophoresis were used to demonstrate RNA quality and quantity. The SpectrumTM and RNeasy® protocols provided the highest quantity and quality of RNA; however, the former revealed superior extraction performance. Consequently, total RNA was extracted from the roots of non-stressed and drought-stressed plants using the SpectrumTM method and six RNA-seq libraries were prepared from polyA + mRNAs by means of TruSeq mRNA library construction protocol to convert RNA to complementary DNA (cDNA). More than 80 million raw read sequences were obtained from each condition with an average read length of 150 bp. The yield and quality of the total RNA were consistently high and the RNA could be used for further analyses as demonstrated by cDNA library construction, RT-PCR, and transcriptome sequencing. Thus, SpectrumTM method can be used to isolate high quality RNA from roots of normal and drought stressed I. paraguariensis plants.Fil: Avico, Edgardo Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; ArgentinaFil: Acevedo, Raúl Maximiliano. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Departamento de Física y Química; ArgentinaFil: Calzadilla, Pablo Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ruiz, Oscar Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Sansberro, Pedro Alfonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentin

Variation of NimC1 expression in Drosophila stocks and transgenic strains.

The NimC1 molecule has been described as a phagocytosis receptor, and is being used as a marker for professional phagocytes, the plasmatocytes, in Drosophila melanogaster. In studies including tumor-biology, developmental biology, and cell mediated immunity, monoclonal antibodies (P1a and P1b) to the NimC1 antigen are used. As we observed that these antibodies did not react with plasmatocytes of several strains and genetic combinations, a molecular analysis was performed on the structure of the nimC1 gene. In these strains we found 2 deletions and an insertion within the nimC1 gene, which may result in the production of a truncated NimC1 protein. The NimC1 positivity was regained by recombining the mutation with a wild-type allele or by using nimC1 mutant lines under heterozygous conditions. By means of these procedures or using the recombined stock, NimC1 can be used as a marker for phagocytic cells in the majority of the possible genetic backgrounds

Broad role for YBX1 in defining the small noncoding RNA composition of exosomes.

RNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their coisolation with contaminants, lack of knowledge of the mechanisms of RNA sorting into EVs, and limitations of conventional RNA-sequencing methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspanin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature transfer RNAs (tRNAs) and other small noncoding RNAs (ncRNAs) encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long ncRNAs, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5' terminal oligopyrimidine (5' TOP) motif. Our results indicate that the RNA-binding protein YBX1, which is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small ncRNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for posttranscriptional modification in the sorting of some RNA species into EVs. Our results suggest that EVs and exosomes could play a role in the purging and intercellular transfer of excess free RNAs, including full-length tRNAs and other small ncRNAs

Plasmodium knowlesi Genome Sequences from Clinical Isolates Reveal Extensive Genomic Dimorphism.

Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology