Genomics in premature infants: A non-invasive strategy to obtain high-quality DNA

We used a cost-effective, non-invasive method to obtain high-quality DNA from buccal epithelial-cells (BEC) of premature infants for genomic analysis. DNAs from BEC were obtained from premature infants with gestational age ≤ 36 weeks. Short terminal repeats (STRs) were performed simultaneously on DNA obtained from the buccal swabs and blood from the same patient. The STR profiles demonstrated that the samples originated from the same individual and exclude any contamination by external DNAs. Whole exome sequencing was performed on DNAs obtained from BEC on premature infants with and without necrotizing enterocolitis, and successfully provided a total number of reads and variants corroborating with those obtained from healthy blood donors. We provide a proof of concept that BEC is a reliable and preferable source of DNA for high-throughput sequencing in premature infants

The Potential Use of Forensic DNA Methods Applied to Sand Fly Blood Meal Analysis to Identify the Infection Reservoirs of Anthroponotic Visceral Leishmaniasis.

BACKGROUND:In the Indian sub-continent, visceral leishmaniasis (VL), also known as kala azar, is a fatal form of leishmaniasis caused by the kinetoplastid parasite Leishmania donovani and transmitted by the sand fly Phlebotomus argentipes. VL is prevalent in northeast India where it is believed to have an exclusive anthroponotic transmission cycle. There are four distinct cohorts of L. donovani exposed individuals who can potentially serve as infection reservoirs: patients with active disease, cured VL cases, patients with post kala azar dermal leishmaniasis (PKDL), and asymptomatic individuals. The relative contribution of each group to sustaining the transmission cycle of VL is not known. METHODOLOGY/PRINCIPAL FINDINGS:To answer this critical epidemiological question, we have addressed the feasibility of an approach that would use forensic DNA methods to recover human DNA profiles from the blood meals of infected sand flies that would then be matched to reference DNA sampled from individuals living or working in the vicinity of the sand fly collections. We found that the ability to obtain readable human DNA fingerprints from sand flies depended entirely on the size of the blood meal and the kinetics of its digestion. Useable profiles were obtained from most flies within the first 24 hours post blood meal (PBM), with a sharp decline at 48 hours and no readable profiles at 72 hours. This early time frame necessitated development of a sensitive, nested-PCR method compatible with detecting L. donovani within a fresh, 24 hours blood meal in flies fed on infected hamsters. CONCLUSION/SIGNIFICANCE:Our findings establish the feasibility of the forensic DNA method to directly trace the human source of an infected blood meal, with constraints imposed by the requirement that the flies be recovered for analysis within 24 hours of their infective feed

ND5 PCR for detection of different levels of <i>L</i>. <i>donovani</i> in <i>P</i>. <i>argentipes</i> flies.

<p>Standard direct PCR (A) or nested PCR (B) reactions on DNA extracted from individual blood fed flies fed on hamsters and mixed with the indicated number of parasites. A-d, flies fed on naïve hamsters as control for primer specificity. EBa, extraction buffer blank for DNA extracted from a-d flies. EBb, extraction blank for DNA extracted from the mixture of flies and parasites. PC1 and NC1 are positive and negative controls for the standard ND5 PCR (A) and the external PCR in the nested reaction (B). Both PC1 and NC1 from the external reaction were cleaned and used as templates for the internal PCR. PC2 and NC2 are positive and negative controls for the second internal nested PCR (B).</p

Total and human DNA extracted from the flies.

<p>Total and human DNA extracted from the flies.</p

Development of a Multiplex Single Base Extension Assay for Mitochondrial DNA Haplogroup Typing

Cilj Razviti test za brzo pretraživanje, kako bi se skratilo vrijeme testiranja i potrošnja uzorka pri određivanju haploskupina mitohondrijske DNA. Postupci U svrhu vrjednovanja testa ukupno je testirano 147 uzoraka, uključujući 73 uzorka kojima je uspješno određena haploskupina s pomoću tipizacije kontrolnog područja (engl. control region, CR) mitohondrijske DNA (mtDNA), 21 uzorak s nepotpuno tipiziranom haploskupinom na osnovi CR, i 31 uzorak poznatog nasljednog izvora kojima prethodno nije tipizirana haploskupina. Dodatno su s pomoću sustava mnogostruke analize kratkih jezgrenih polimorfnih odsječaka (engl., short nuclear polymorphism, SNP) analizirane dvije jako degradirane ljudske kosti zakopane krajem četrdeseetih godina prošloga stoljeća. Rezultati Kad je mnogostruki sustav SNP primijenjen za tipizaciju 96 uzoraka s prethodno sekvencioniranim CR, opaženo je povećanje definiranja haploskupine ili makrohaploskupine u odnosu na uobičajenu analizu slijeda CR. Prošireni test za pojedinačne baze uspješno je primijenjen u određivanju haploskupina iz uzoraka kostiju starih desetljećima, još iz II. Svjetskoga rata. Zaključak Mnogostruki sustav SNP uspješno je primijenjen za određivanje statusa haploskupina jako raspadnutih ljudskih kostiju, čime je pokazao sposobnost odstranjenja mogućih onečišćenja. Mnogostruki sustav SNP predstavlja jeftin a vrlo učinkovit postupak kojim se mogu tipizirati haploskupine mtDNA A, B, C, D, E, F, G, H, L1/L2, L3, M i N, što znači da se može dobro iskoristiti za brza pretraživanja pri identifikaciji ljudi ili u antropološkim istraživanjima.Aim To provide a screening tool to reduce time and sample consumption when attempting mitochondrial DNA (mtDNA) haplogroup typing. Methods A single base primer extension assay was developed to enable typing, in a single reaction, of twelve mtDNA haplogroup specific polymorphisms. For validation purposes a total of 147 samples were tested including 73 samples successfully haplogroup typed using mtDNA control region (CR) sequence data, 20 samples inconclusively haplogroup typed by CR sequence data, 21 samples previously haplogroup typed using restriction fragment length polymorphism (RFLP) analysis, and 31 samples of known ancestral origin without previous haplogroup typing. Additionally, two highly degraded human bones embalmed and buried in the early 1950s were analyzed using the single nucleotide polymorphisms (SNP) multiplex. Results When the SNP multiplex was used to type the 96 previously CR sequenced specimens, an increase in haplogroup or macrohaplogroup assignment relative to conventional CR sequence analysis was observed. The single base extension assay was also successfully used to assign a haplogroup to decades-old, embalmed skeletal remains dating to World War II. Conclusion The SNP multiplex was successfully used to obtain haplogroup status of highly degraded human bones, and demonstrated the ability to eliminate possible contributors. The SNP multiplex provides a low-cost, high throughput method for typing of mtDNA haplogroups A, B, C, D, E, F, G, H, L1/L2, L3, M, and N that could be useful for screening purposes for human identification efforts and anthropological studies

Allele frequency calculations in flies yielding usable profiles.

<p>Allele frequency calculations in flies yielding usable profiles.</p

The kinetics of blood meal digestion determines the time frame for detecting human DNA.

<p>(A) Photomicrographs depicting flies with a full or partial blood meal at day 0, and the kinetics of blood meal digestion on days 1–3 PBM. (B) Total and human DNA at days 0, 1, 2, 3 and 5 PBM. The results indicate the mean value of eight different flies, four from each subject ±SD. (C) The percentage of flies that yielded a STR profile suitable for comparison out of eight flies tested on each day.</p

Detection of <i>L</i>. <i>donovani</i> amastigotes in a fresh blood meal.

<p>Two independent experiments (A and B) showing nested PCR amplification of ND5 gene from flies fed on infected (i) or naïve (ii) hamsters. Arrows indicate flies showing positive reaction. EB, DNA extraction blank; PC, positive control for the first, external PCR (PC1) or the second internal fragment (PC2). NC, negative control for the first, external (NC1) or the second, internal (NC2) PCR reactions. The faint bands observed above the primers at lanes 11 and 13 in panel B ii are not at the same size of the true PCR product and are most likely a product of primer dimerization.</p

The effect of storage conditions on the integrity of human DNA.

<p>The effect of storage conditions on the integrity of human DNA.</p