The Impact of DNA Polymerase and Number of Rounds of Amplification in PCR on 16S rRNA Gene Sequence Data

Sze, Marc A.; Schloss, Patrick D.

doi:10.1128/msphere.00163-19

Cited by 106 publications

(78 citation statements)

References 52 publications

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“…The PCR steps are also optimized for the same purpose, minimizing amplification cycles and stopping before most reactions plateau. Using fewer PCR steps, we decrease error rates and chimera formation, as previously reported 18,21 . The agarose gel check after library amplifications can still be useful for samples with sufficient biomass, though it is often the case that low biomass samples show no visible bands, hampering any useful interpretation 12 .…”

Section: Equivolumetric Protocol For Amplicon Library Preparationsupporting

confidence: 57%

Equivolumetric protocol generates library sizes proportional to total microbial load in next-generation sequencing

Gnf¹,

Ap²,

Lfv³

2020

Preprint

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Next-generation sequencing (NGS) has been extensively employed to perform microbiome characterization worldwide. As a culture-independent methodology, it has allowed high-level profiling of sample microbial composition. However, most studies are limited to information regarding relative bacterial abundances, ignoring scenarios in which sample microbe biomass can vary widely. Here, we develop an equivolumetric protocol for amplicon library preparation capable of generating NGS data responsive to input DNA, recovering proportionality between observed read counts and absolute bacterial abundances. Under specified conditions, we argue that the estimation of colony-forming units (CFU), the most common unit of bacterial abundance in classical microbiology, is challenged mostly by resolution and taxon-to-taxon variation. We propose Bayesian cumulative probability models to address such issues. Our results indicate that predictive errors vary consistently below one order of magnitude for observed bacteria. We also demonstrate our approach has the potential to generalize to previously unseen bacteria, but predictive performance is hampered by specific taxa of uncommon profile. Finally, it remains clear that NGS data are not inherently restricted to relative information only, and microbiome science can indeed meet the working scales of traditional microbiology.

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Section: Equivolumetric Protocol For Amplicon Library Preparationsupporting

confidence: 57%

Equivolumetric protocol generates library sizes proportional to total microbial load in next-generation sequencing

Gnf¹,

Ap²,

Lfv³

2020

Preprint

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“…This has been done by creating sequence concatenates, for example using rolling circular amplification, [4, 5, 6] or via gene barcoding prior to amplification [7]. These methods usually reduce the number of different variants that can be studied, because a part of minION throughput is invested in reading duplicated reads, and is sensitive to bias occuring during the amplification steps [8]. Other approaches, used for genome assembly, combine long minION reads with shorter and more accurate reads obtained via other technologies, such as Illumina sequencing-by-synthesis approach [9].…”

Section: Introductionmentioning

confidence: 99%

SINGLe: Accurate detection of single nucleotide polymorphisms using nanopore sequencing in gene libraries

Espada

Zarevski

Dramé-Maigné

et al. 2020

Preprint

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words)Background Nanopore sequencing is a powerful single 1 molecule DNA sequencing technology which provides a 2 high throughput and long sequence reads. Nevertheless, 3 its relatively high native error rate limits the direct detec-4 tion of point mutations in individual reads of amplicon li-5 braries, as these mutations are difficult to distinguish from 6 the sequencing noise. 7 Results We propose a computational method to reduce 8 noise in nanopore detection of point variations. Our ap-9 proach uses the fact that all reads are expected to be 10 very similar to a wild type sequence, for which we ex-11 perimentally characterize the position-specific systematic 12 sequencing error pattern. We then use this information to 13 reweight, in individual reads from the variant library, the 14 confidence given to nucleotides read that do not match the 15 wild type. We tested this method on two sets of known 16 variants of Klen Taq, where the true mutation rate was 17 3.3 mutations per kb, well below the sequencing noise. We 18 observed that the actual mutations became more distin-19 guishable from sequencing noise after correction. This ap-20 proach can be used, for example to help the clustering of 21 variants, or to decrease the number of reads necessary to 22 call a consensus. 23Conclusions The computational method is simple to im-24 plement and requires only a few thousands reads of the 25 wild type sequence of interest, which can be easily ob-26 tained by multiplexing in a single minION run. The ap-27 proach does not require any modification in the experimen-28 tal protocol for sequencing and can be simply implemented 29 downstream standard base calling.30 Keywords 31 minION, nanopore sequencing, next generation sequenc-32 ing, amplicons, SNP detection, logistic regression.33In this paper, we propose a computational protocol to im-74 prove variant detection in individual reads from libraries 75 for which a reference gene is known, using standard 1D 76 protocol minION sequencing. We base our method on two 77 observations made during the sequencing of many (identi-78 cal) copies of the parent sequence. First, the confidence or 79 quality scores (Q score ) assigned by the base calling process 80 to each nucleotide are usually low when a wrong nucleotide 81 is assigned (Suppl. figure S2), as expected. Second, the er-82 rors are not homogeneously distributed, and they are more 83 frequent in some positions of the DNA (Suppl. figure S1). 84These observations suggest that it should be possible to re-85 duce the non-random part of the sequencing errors, using 86 the information contained in the (Q score ). The method we 87 propose has two steps: the first one uses the reference reads 88 to build a statistical model of the error pattern. Here we 89 used a position and nucleotide-specific logistic regression. 90In the second step, this information is used to re-analyze 91 minION base calls for the variant library and to update the 92 confidence value of each nucleotide read in this dataset. 93 We tested our method us...

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“…Nevertheless, metagenomic methods are known to be prone to errors at different steps of the work ow, from sample collection [31][32][33][34], DNA extraction [35,36], library preparation and sequencing [37,38] to data analysis [39,40]. In order to facilitate the implementation of these methods into clinical routine practice, standardized methods are urgently needed [41][42][43][44][45].…”

Section: Introductionmentioning

confidence: 99%

A unique and reliable fecal DNA extraction method for 16S rRNA gene and shotgun metagenomic sequencing in the analysis of the human gut microbiome

Elie¹,

Perret²,

Louis³

et al. 2020

Preprint

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Background: The gut microbiome is widely analyzed using high-throughput sequencing, such as 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing (SMS). DNA extraction is known to have a large impact on the metagenomic analyses. The aim of this study was to select a unique and best performing DNA extraction protocol for both metagenomic sequencing methods. In that context, four commonly used DNA extraction methods were compared for the analysis of the gut microbiota. Commercial versions were evaluated against modified protocols using a stool preprocessing device (SPD, bioMérieux) in order to facilitate DNA extraction. Stool samples from nine healthy volunteers and nine patients with a Clostridium difficile infection were extracted with all protocols and sequenced with both metagenomic methods. Protocols were ranked using wet- and dry-lab criteria, including quality controls of the extracted genomic DNA, alpha-diversity, accuracy using a mock community of known composition and repeatability across technical replicates.Results: Independently of the sequencing methods used, SPD significantly improved efficiency of the four tested protocols compared with their commercial version, in terms of extracted DNA quality, accuracy of the predicted composition of the microbiota (notably for Gram-positive bacteria), sample alpha-diversity, and experimental repeatability. The best overall performance was obtained for the S-DQ protocol, SPD combined to the DNeasy PowerLyser PowerSoil protocol from QIAGEN.Conclusion: Based on this evaluation, we recommend to use the S-DQ protocol, to obtain standardized and high quality extracted DNA in the human gut microbiome studies.

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The Impact of DNA Polymerase and Number of Rounds of Amplification in PCR on 16S rRNA Gene Sequence Data

Cited by 106 publications

References 52 publications

Equivolumetric protocol generates library sizes proportional to total microbial load in next-generation sequencing

Equivolumetric protocol generates library sizes proportional to total microbial load in next-generation sequencing

SINGLe: Accurate detection of single nucleotide polymorphisms using nanopore sequencing in gene libraries

A unique and reliable fecal DNA extraction method for 16S rRNA gene and shotgun metagenomic sequencing in the analysis of the human gut microbiome

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