Naslov
Comparing effectiveness of DNA extraction kits and assessing relative abundances of microbial phyla in human fecal specimens by two next-generation sequencing platforms: key methodological steps of the 􀈤MINUTE for IBD􀈥 project from Croatia

Autori
Meštrović Tomislav ; Čipčić Paljetak Hana ; Perić Mihaela ; Panek Marina ; Matijašić Mario ; Vranešić Bender Darija ; Kunović Ana ; Krznarić Željko ; Verbanac Donatella

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
ASM Microbe

Mjesto i datum
New Orleans (LA), Sjedinjene Američke Države, 01.06.2017. - 05.06.2017

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
human faecal samples, DNA extraction kits, next-generation sequencing, gut microbiota composition

Sažetak
The translational project “Assessment of Microbiota, Inflammatory Markers, Nutritional and Endocrinological Status in IBD Patients (MINUTE for IBD)” is the first Croatian microbiome research endeavor to explore host-gut microbiota interactions, with an end-goal of defining novel approaches for the management of inflammatory bowel disease. As the results and final output may be highly influenced by methodological issues (from sample collection and extraction to sequencing approach), our initial aims were to evaluate DNA yield and quality of different DNA extraction kits, as well as to compare the sequencing results by two different next-generation sequencing (NGS) platforms. Fecal samples were collected fresh and in OMNIgene.GUT device from healthy donors and then stored for 14 days at -20°C (-4 °F) and at room temperature, respectively. Three commercially available DNA extraction kits were used (QIAamp Fast DNA Stool Mini Kit, MO BIO PowerFecal DNA Isolation Kit and MP Biomedicals Fast DNA SPIN Kit for Feces). Gel electrophoresis was employed to test sample integrity, whereas Qubit fluorometer was used to verify sample purity and concentration. Fecal bacterial communities were profiled by 16S rRNA amplicon sequencing using two NGS platforms – Illumina MiSeq platform (targeting V3 and V4 hypervariable regions) and Ion Torrent PGM System (targeting hypervariable regions V2-V9). Bioinformatic analysis of sequence data was performed by using QIIME. The quality and yield of DNA varied between DNA extraction kits, with MP Biomedicals showing optimal results, followed by QIAamp and MO BIO kits. Although the donor-specific bacterial diversity was not influenced, we demonstrated kit-specific abundance levels among the most prevalent bacterial families (most notably Bacteroidaceae and Lachnospiraceae). Further, when comparing the sequencing results by two NGS platforms, comparable relative abundance ratios were observed for eight of the ten most abundant phyla (i.e. Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Cyanobacteria, Tenericutes, Lentisphaerae and Acidobacteria) ; the exception were phyla Fusobacteria and Verrucomicrobia, represented in higher abundance when using Illumina MiSeq platform. The composition of donor-specific microbiota is generally maintained irrespective of the DNA extraction kit and NGS platform used, albeit certain differences that were observed may be relevant in choosing the appropriate technology for specific research questions requests.

Izvorni jezik
Engleski

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