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Application of different DNA extraction procedures, library preparation protocols and sequencing platforms: impact on sequencing results.

ABSTRACT: In this study three DNA extraction procedures, two library preparation protocols and two sequencing platforms were applied to analyse six bacterial cultures and their corresponding DNA obtained as part of a proficiency test. The impact of each variable on sequencing results was assessed using the following parameters: reads quality, assembly and alignment statistics; number of single nucleotide polymorphisms (SNPs), detected applying assembly- and alignment-based strategies; antimicrobial resistance genes (ARGs), identified on de novo assemblies of all sequenced genomes. The investigated nucleic acid extraction procedures, library preparation kits and sequencing platforms do not significantly affect de novo assembly statistics and number of SNPs and ARGs. The only exception was observed for two duplicates, which were associated to one PCR-based library preparation kit. Results from this comparative study can support researchers in the choice toward the available pre-sequencing and sequencing options, and might suggest further comparisons to be performed.

SUBMITTER: Pasquali F

PROVIDER: S-EPMC6838873 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Application of different DNA extraction procedures, library preparation protocols and sequencing platforms: impact on sequencing results.

Pasquali F F Do Valle I I Palma F F Remondini D D Manfreda G G Castellani G G Hendriksen R S RS De Cesare A A

Heliyon 20191001 10

In this study three DNA extraction procedures, two library preparation protocols and two sequencing platforms were applied to analyse six bacterial cultures and their corresponding DNA obtained as part of a proficiency test. The impact of each variable on sequencing results was assessed using the following parameters: reads quality, assembly and alignment statistics; number of single nucleotide polymorphisms (SNPs), detected applying assembly- and alignment-based strategies; antimicrobial resist ...[more]

PMID: 31720479

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Project description:BackgroundMicroRNAs(miRNAs) are 18-25 nt small RNAs playing critical roles in many biological processes. The majority of known miRNAs were discovered by conventional cloning and a Sanger sequencing approach. The next-generation sequencing (NGS) technologies enable in-depth characterization of the global repertoire of miRNAs, and different protocols for miRNA library construction have been developed. However, the possible bias between the relative expression levels and sequences introduced by different protocols of library preparation have rarely been explored.ResultsWe assessed three different miRNA library preparation protocols, SOLiD, Illumina versions 1 and 1.5, using cloning or SBS sequencing of total RNA samples extracted from skeletal muscles from Hu sheep and Dorper sheep, and then validated 9 miRNAs by qRT-PCR. Our results show that SBS sequencing data highly correlate with Illumina cloning data. The SOLiD data, when compared to Illumina's, indicate more dispersed distribution of length, higher frequency variation for nucleotides near the 3'- and 5'-ends, higher frequency occurrence for reads containing end secondary structure (ESS), and higher frequency for reads that do not map to known miRNAs. qRT-PCR results showed the best correlation with SOLiD cloning data. Fold difference of Hu sheep and Dorper sheep between qRT-PCR result and SBS sequencing data correlated well (r = 0.937), and fold difference of miR-1 and miR-206 among SOLiD cloning data, qRT-PCR and SBS sequencing data was similar.ConclusionsThe sequencing depth can influence the quantitative measurement of miRNA abundance, but the discrepancy caused by it was not statistically significant as high correlation was observed between Illumina cloning and SBS sequencing data. Bias of length distribution, sequence variation, and ESS was observed between data obtained with the different protocols. SOLiD cloning data differ from Illumina cloning data mainly because of distinct methods of adapter ligation. The good correlation between qRT-PCR result and SOLiD data might be due to the similarities of the hybridization-based methods. The fold difference analysis indicated that methods based on hybridization may be superior for quantitative measurement of miRNA abundance. Because of the genome sequence of the sheep is not available, our data may not explain how the entire miRNA bias in the natural miRNAs in sheep or other mammal miRNA expression, unbiased artificially synthesized miRNA will help on evaluating the methodology of miRNA library preparation.

Project description:Whole genome sequencing (WGS) has become the new gold standard for bacterial outbreak investigation, due to the high resolution available for typing. While sequencing is currently predominantly performed on Illumina devices, the preceding library preparation can be performed using various protocols. Enzymatic fragmentation library preparation protocols are fast, have minimal hands-on time, and work with small quantities of DNA. The aim of our study was to compare three library preparation protocols for molecular typing: Nextera XT (Illumina); Nextera Flex (Illumina); and QIAseq FX (Qiagen). We selected 12 ATCC strains from human Gram-positive and Gram-negative pathogens with %G+C-content ranging from 27% (Fusobacterium nucleatum) to 73% (Micrococcus luteus), each having a high quality complete genome assembly available, to allow in-depth analysis of the resulting Illumina sequence data quality. Additionally, we selected isolates from previously analyzed cases of vancomycin-resistant Enterococcus faecium (VRE) (n = 7) and a local outbreak of Klebsiella aerogenes (n = 5). The number of protocol steps and time required were compared, in order to test the suitability for routine laboratory work. Data analyses were performed with standard tools commonly used in outbreak situations: Ridom SeqSphere+ for cgMLST; CLC genomics workbench for SNP analysis; and open source programs. Nextera Flex and QIAseq FX were found to be less sensitive than Nextera XT to variable %G+C-content, resulting in an almost uniform distribution of read-depth. Therefore, low coverage regions are reduced to a minimum resulting in a more complete representation of the genome. Thus, with these two protocols, more alleles were detected in the cgMLST analysis, producing a higher resolution of closely related isolates. Furthermore, they result in a more complete representation of accessory genes. In particular, the high data quality and relative simplicity of the workflow of Nextera Flex stood out in this comparison. This thorough comparison within an ISO/IEC 17025 accredited environment will be of interest to those aiming to optimize their clinical microbiological genome sequencing.

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Application of different DNA extraction procedures, library preparation protocols and sequencing platforms: impact on sequencing results.

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Application of different DNA extraction procedures, library preparation protocols and sequencing platforms: impact on sequencing results.

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