Comparison of reduced metagenome and 16S rRNA gene sequencing for determination of genetic diversity and mother-child overlap of the gut associated microbiota

Ray, Anuradha; Avershina, Ekaterina; Angell, Inga Leena; Ludvigsen, Jane; Manohar, Prasanth; Padmanaban, Sumathi; Nachimuthu, Ramesh; Snipen, Lars; Rudi, Knut

doi:10.1016/j.mimet.2018.02.016

Cited by 25 publications

(31 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Reduced representation sequencing of metagenome samples using restriction enzyme has previously been shown to perform well compared to 16S and metagenome shotgun sequencing [39][40][41]. While our study used a single digest of ApeKI or PstI, these studies all used a double digest (i.e.…”

Section: Utility Of a High-throughput Metagenomics Methodsmentioning

confidence: 99%

A restriction enzyme reduced representation sequencing approach for low-cost, high-throughput metagenome profiling

et al. 2020

View full text Add to dashboard Cite

Microbial community profiles have been associated with a variety of traits, including methane emissions in livestock. These profiles can be difficult and expensive to obtain for thousands of samples (e.g. for accurate association of microbial profiles with traits), therefore the objective of this work was to develop a low-cost, high-throughput approach to capture the diversity of the rumen microbiome. Restriction enzyme reduced representation sequencing (RE-RRS) using ApeKI or PstI, and two bioinformatic pipelines (referencebased and reference-free) were compared to bacterial 16S rRNA gene sequencing using repeated samples collected two weeks apart from 118 sheep that were phenotypically extreme (60 high and 58 low) for methane emitted per kg dry matter intake (n = 236). DNA was extracted from freeze-dried rumen samples using a phenol chloroform and bead-beating protocol prior to RE-RRS. The resulting sequences were used to investigate the repeatability of the rumen microbial community profiles, the effect of laboratory and analytical method, and the relationship with methane production. The results suggested that the best method was PstI RE-RRS analyzed with the reference-free approach, which accounted for 53.3±5.9% of reads, and had repeatabilities of 0.49±0.07 and 0.50±0.07 for the first two principal components (PC1 and PC2), phenotypic correlations with methane yield of 0.43±0.06 and 0.46±0.06 for PC1 and PC2, and explained 41±8% of the variation in methane yield. These results were significantly better than for bacterial 16S rRNA gene sequencing of the same samples (p<0.05) except for the correlation between PC2 and methane yield. A Sensitivity study suggested approximately 2000 samples could be sequenced in a single lane on an Illumina HiSeq 2500, meaning the current work using 118 samples/lane and future proposed 384 samples/lane are well within that threshold. With minor adaptations, our approach could be used to obtain microbial profiles from other metagenomic samples.

show abstract

Section: Utility Of a High-throughput Metagenomics Methodsmentioning

confidence: 99%

A restriction enzyme reduced representation sequencing approach for low-cost, high-throughput metagenome profiling

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Previous use of ddRADseq have reported biases in the signals, most likely introduced in the PCR amplification of such fragments [16]. To investigate this we used mock data from [8], where some samples included a single genome only. In Figure 4 we have plotted the data from four such samples.…”

Section: Resultsmentioning

confidence: 99%

“…All these 9 mock mixtures where done in triplicates, resulting in 27 samples. All samples were subject to the wet-lab procedures described in [8] to obtain paired-end Illumina HiSeq reads. The restriction enzymes EcoRI and MseI were used throughout this study.…”

Section: Mock Datamentioning

confidence: 99%

“…As an alternative to do a full shotgun metagenomic sequencing, the use of restriction enzymes to reduce the genomic sequence space has also been employed to investigate microbial communities [7,8,9]. The double-digested restriction associated DNA sequencing (ddRADseq) idea [10,11,12] has been along for some time, but its use for metagenome studies is quite new.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, the approach has been termed Reduced Metagenome Sequencing (RMS). The wet-lab protocols for ddRADseq are well established and straightforward [8].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Reduced Metagenome Sequencing for strain-resolution taxonomic proles

Snipen

Angell

Rognes

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Background: Studies of shifts in microbial community composition has many applications. For studies at species or subspecies levels, the 16S amplicon sequencing lacks resolution, and is often replaced by full shotgun sequencing. Due to higher costs, this restricts the number of samples sequenced. As an alternative to a full shotgun sequencing we have investigated the use of Reduced Metagenome Sequencing (RMS) to estimate the composition of a microbial community. This involves the use of double-digested restriction associated DNA sequencing, which means only a smaller fraction of the genomes are sequenced. The read sets obtained by this approach have properties different from both amplicon and shotgun data, and analysis pipelines for both can either not be used at all or do not explore the full potential of RMS data.Results: We suggest a procedure for analyzing such data, based on fragment clustering and the use of a constrained ordinary least square de-convolution for estimating the relative abundance of all community members. Mock-community data sets shows the potential to clearly separate between strains even when the 16S is 100% identical and genome-wide dierences is < 0:02, indicating RMS has a very high resolution. From a simulation study we compare RMS to shotgun sequencing and show that we get improved abundance estimates when the community has many very closely related genomes. From a real data set of infants guts we show that RMS is capable of detecting a strain-diversity gradient for Escherichia coli across time.Conclusion: We find that RMS is a good alternative to either metabarcoding or shotgun sequencing when it comes to resolving microbial communities at the strain-level. Like shotgun metagenomics, it requires a good database of reference genomes, and is well suited for studies of the human gut or other communities where many reference genomes exist. A data analysis pipeline is offered, as an R package at https://github.com/larssnip/microRMS.

show abstract

The neonatal intestinal resistome and factors that influence it—a systematic review

Leo

Curtis

Zimmermann

2022

Clinical Microbiology and Infection

View full text Add to dashboard Cite

Comparison of reduced metagenome and 16S rRNA gene sequencing for determination of genetic diversity and mother-child overlap of the gut associated microbiota

Cited by 25 publications

References 25 publications

A restriction enzyme reduced representation sequencing approach for low-cost, high-throughput metagenome profiling

A restriction enzyme reduced representation sequencing approach for low-cost, high-throughput metagenome profiling

Reduced Metagenome Sequencing for strain-resolution taxonomic proles

The neonatal intestinal resistome and factors that influence it—a systematic review

Contact Info

Product

Resources

About