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

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