Optimizing PCR primers targeting the bacterial 16S ribosomal RNA gene

Sambo, Francesco; Finotello, Francesca; Lavezzo, Enrico; Baruzzo, Giacomo; Masi, Giulia; Peta, Elektra; Falda, Marco; Toppo, Stefano; Barzon, Luisa; Camillo, Barbara Di

doi:10.1186/s12859-018-2360-6

Cited by 56 publications

(45 citation statements)

References 32 publications

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“…While human contamination is a very common problem in amplification-free shotgun metagenomic sequencing strategies 22 , it is under reported as an issue for 16S rRNA gene sequencing, due to the use of bacteria/archaea specific primers. However, degenerate primers are routinely used for 16S rRNA sequencing 23 . This increases coverage, in terms of the number of 16S rRNA sequences matched by at least one primer, but also allows for off target amplification of non-bacterial DNA.…”

Section: Resultsmentioning

confidence: 99%

Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis

Walker

Barrett

Hogan

et al. 2020

Sci Rep

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The targeted sequencing of the 16S rRNA gene is one of the most frequently employed techniques in the field of microbial ecology, with the bacterial communities of a wide variety of niches in the human body have been characterised in this way. This is performed by targeting one or more hypervariable (V) regions within the 16S rRNA gene in order to produce an amplicon suitable in size for next generation sequencing. To date, all technical research has focused on the ability of different V regions to accurately resolve the composition of bacterial communities. We present here an underreported artefact associated with 16S rRNA gene sequencing, namely the off-target amplification of human DNA. By analysing 16S rRNA gene sequencing data from a selection of human sites we highlighted samples susceptible to this off-target amplification when using the popular primer pair targeting the V3–V4 region of the gene. The most severely affected sample type identified (breast tumour samples) were then re-analysed using the V1–V2 primer set, showing considerable reduction in off target amplification. Our data indicate that human biopsy samples should preferably be amplified using primers targeting the V1–V2 region. It is shown here that these primers result in on average 80% less human genome aligning reads, allowing for more statistically significant analysis of the bacterial communities residing in these samples.

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Section: Resultsmentioning

confidence: 99%

Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis

Walker

Barrett

Hogan

et al. 2020

Sci Rep

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“…The data presented warrants that additional research should be conducted on optimizing the sequences employed during use of the 16S rRNA encoding gene as there appears to be a certain lack of discriminatory power as reported by authors. The 16S rRNA gene has been noted to be omnipresent in all prokaryotes and it contains both fast-evolving and slow-evolving regions (Sambo et al, 2018). As such, it is possible to design broad-spectrum primer pairs targeting the slow-evolving regions to obtain a PCR product and subsequently sequence information from the generated amplicon can be utilized to isolate out the speciesspecific fast-evolving regions hence contributing to an overall increase in discriminatory power (Sambo et al, 2018).…”

Section: Polymerase Chain Reaction (Pcr) Methodsmentioning

confidence: 99%

“…The 16S rRNA gene has been noted to be omnipresent in all prokaryotes and it contains both fast-evolving and slow-evolving regions (Sambo et al, 2018). As such, it is possible to design broad-spectrum primer pairs targeting the slow-evolving regions to obtain a PCR product and subsequently sequence information from the generated amplicon can be utilized to isolate out the speciesspecific fast-evolving regions hence contributing to an overall increase in discriminatory power (Sambo et al, 2018). The key challenge of using the 23S rRNA encoding gene would be the dearth of established broad-range bacterial PCR amplification and sequencing primers and hence the solution would be to develop novel PCR assays targeting the said gene (Hunt et al, 2006).…”

Section: Polymerase Chain Reaction (Pcr) Methodsmentioning

confidence: 99%

An update on the detection methods of Parachlamydia acanthamoebae, an atypical agent of pneumonia

Rames

2019

APJMBB

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Parachlamydia acanthamoebae (P. acanthamoebae) has been recognized as an emerging agent of pneumonia as it has been identified in human samples via culture-based, molecular and serological techniques. Additionally, studies on animal models have shown that it fulfills the third and fourth Koch postulates to be assigned a pathogenic role. Due to the threat posed by it, multiple tools have been employed in the search for P. acanthamoebae. The methods utilized for its detection would be cell culture based approaches which involve both animal and amoebal cell culture and also molecular techniques that encompasses polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and in situ hybridization (ISH). Additionally, immunohistochemistry (IHC) and serology based techniques such as direct and indirect immunofluorescence are also employed with the usage of Western blotting or immunoblotting as confirmatory procedures. This review attempts to describe the variety of techniques that are present in literature for the isolation and identification of P. acanthamoebae.

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“…A wide range of (proposed) universal PCR primer pairs for amplification of a particular hypervariable region of the 16S rRNA gene have been designed for achieving accurate metagenomic profiling [8,18,19]. Nevertheless, due to their variable amplification performances, being caused by inconsistent sequence complementarity with corresponding bacterial 16S rRNA sequences, and variable discriminatory power of the amplified hypervariable region, no single PCR primer pair has become the gold standard for 16S rRNA gene-based microbial profiling.…”

Section: Selection Of Publicly Available 16s Rrna Gene-based Primer Pmentioning

confidence: 99%

The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

et al. 2020

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Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.

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Optimizing PCR primers targeting the bacterial 16S ribosomal RNA gene

Cited by 56 publications

References 32 publications

Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis

Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis

An update on the detection methods of Parachlamydia acanthamoebae, an atypical agent of pneumonia

The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

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