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Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity.

ABSTRACT: Background:Genomic testing is often limited by the exhaustible nature of human tissue and blood samples. Here we describe biotinylated amplicon sequencing (BAmSeq), a method that allows for the creation of PCR amplicon based next-generation sequencing (NGS) libraries while retaining the original source DNA. Design and methods:Biotinylated primers for different loci were designed to create NGS libraries using human genomic DNA from cell lines, plasma, and formalin-fixed paraffin embedded (FFPE) tissues using the BAmSeq protocol. DNA from the original template used for each BAmSeq library was recovered after separation with streptavidin magnetic beads. The recovered DNA was then used for end-point, quantitative and droplet digital PCR (ddPCR) as well as NGS using a cancer gene panel. Results:Recovered DNA was analyzed and compared to the original DNA after one or two rounds of BAmSeq. Recovered DNA revealed comparable genomic distributions and mutational allelic frequencies when compared to original source DNA. Sufficient quantities of recovered DNA after BAmSeq were obtained, allowing for additional downstream applications. Conclusions:We demonstrate that BAmSeq allows original DNA template to be recovered with comparable quality and quantity to the source DNA. This recovered DNA is suitable for many downstream applications and may prevent sample exhaustion, especially when DNA quantity or source material is limiting.

SUBMITTER: Cravero K

PROVIDER: S-EPMC6104457 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity.

Cravero Karen K Medford Arielle A Pallavajjala Aparna A Canzoniero Jenna J Hunter Natasha N Chu David D Cochran Rory L RL Waters Ian I Christenson Eric S ES Kyker-Snowman Kelly K Button Berry B Cole Alex J AJ Park Ben Ho BH

Practical laboratory medicine 20180809

<h4>Background</h4>Genomic testing is often limited by the exhaustible nature of human tissue and blood samples. Here we describe biotinylated amplicon sequencing (BAmSeq), a method that allows for the creation of PCR amplicon based next-generation sequencing (NGS) libraries while retaining the original source DNA.<h4>Design and methods</h4>Biotinylated primers for different loci were designed to create NGS libraries using human genomic DNA from cell lines, plasma, and formalin-fixed paraffin em ...[more]

PMID: 30140723

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Project description:BACKGROUND:High-throughput sequencing techniques are used to analyse the diversity of the respiratory microbiota in health and disease. Although extensive data are available regarding bacterial respiratory microbiota, its fungal component remains poorly studied. This is partly due to the technical issues associated with fungal metagenomics analyses. In this study, we compared two DNA extraction protocols and two fungal amplification targets for combined bacterial and fungal targeted amplicon sequencing analyses of the respiratory microbiota. METHODS:Six sputa, randomly selected from routine samples in Mondor Hospital (Creteil, France) and treated anonymously, were tested after bacterial and fungal routine culture. Two of which were spiked with Aspergillus Fumigati and Aspergillus Nigri (105 conidia/mL). After mechanical lysis, DNA was extracted using automated QIAsymphony® extraction (AQE) or manual PowerSoil® MoBio extraction (MPE). DNA yield and purity were compared. DNA extracted from spiked sputa was subjected to (i) real-time PCR for Aspergillus DNA detection and (ii) combined metagenomic analyses targeting barcoded primers for fungal ITS1 and ITS2, and bacterial V1-V2 and V3-V4 16S regions. Amplicon libraries were prepared using MiSeq Reagent V3 kit on Illumina platform. Data were analysed using PyroMIC© and SHAMAN software, and compared with culture results. RESULTS:AQE extraction provided a higher yield of DNA (AQE/MPE DNA ratio = 4.5 [1.3-11]) in a shorter time. The yield of Aspergillus DNA detected by qPCR was similar for spiked sputa regardless of extraction protocol. The extraction moderately impacted the diversity or relative abundances of bacterial communities using targeted amplicon sequencing (2/43 taxa impacted). For fungi, the relative abundances of 4/11 major taxa were impacted and AQE results were closer to culture results. The V1-V2 or V3-V4 and ITS1 or ITS2 targets assessed similarly the diversity of bacterial and fungal major taxa, but ITS2 and V3-V4 detected more minor taxa. CONCLUSION:Our results showed the importance of DNA extraction for combined bacterial and fungal targeted metagenomics of respiratory samples. The extraction protocol can affect DNA yield and the relative abundances of few bacterial but more fungal taxa. For fungal analysis, ITS2 allowed the detection of a greater number of minor taxa compared with ITS1.

Project description:Bovine mycotic abortion is sporadic and caused by different ubiquitous and opportunistic fungi. Recently, a broad spectrum of bacterial opportunists involved in bovine abortion was revealed by 16S rRNA gene amplicon sequencing. We hypothesized that fungal organisms potentially involved in bovine abortion also might remain undetected by conventional culture. In this retrospective study, we therefore applied fungal internal transcribed spacer 2 (ITS2) region amplicon sequencing to 74 cases of bovine abortion submitted to our diagnostic service. The investigation was complemented by fungal culture and, retrospectively, by data from bacteriological, virological and parasitological analyses and histopathological examination of placentas. Fungal DNA was found in both the placentas and abomasal contents, with 92 fungal genera identified. In 18 cases, >75% of the reads belonged to one specific fungal genus: Candida (n = 7), Malassezia (n = 4), Cryptococcus (n = 3), unidentified Capnodiales (n = 3), Actinomucor (n = 1), Cystofilobasidium (n = 1), Penicillium (n = 1), Verticillum (n = 1) and Zymoseptoria (n = 1) with one case harboring two different genera. By culture, in contrast, fungal agents were detected in only 6 cases. Inflammatory and/or necrotizing lesions were found in 27/40 histologically assessed placentas. However, no lesion-associated fungal structures were detected in HE- and PAS-stained specimens. Complementary data revealed the presence of one or more non-fungal possible abortifacient: Chlamydiales, Coxiella burnetii, Leptospira spp., Campylobacter fetus subsp. fetus, Streptococcus uberis, Escherichia coli, Streptococcus pluranimalium, Bacillus licheniformis, Campylobacter fetus subsp. fetus, Serratia marcescens, Trueperella pyogenes, Schmallenbergvirus, Neospora caninum. The mycobiota revealed by sequencing did not differ between cases with or without a possible infectious etiology. Our study suggests that amplicon sequencing of the ITS2 region from DNA isolated from bovine abortion does not provide additional information or new insight into mycotic abortion and without complementary analyses may easily lead to a false interpretation of the role of fungal organisms in bovine abortion.

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Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity.

Publications

Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity.

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