Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis

Danhorn, Thomas; Young, Charles W.; DeLong, Edward F.

doi:10.1038/ismej.2012.35

Cited by 22 publications

(22 citation statements)

References 43 publications

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“…Replication is fundamental to rigorous experimental design [25], but it is only now becoming financially possible for metagenomic studies [26,27]. Here we examined replicate metagenomes across varied DNA input amounts, library preparation procedures, and sequencing platforms.…”

Section: Discussionmentioning

confidence: 99%

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Sequencing platform and library preparation choices impact viral metagenomes

Solonenko

Ignacio‐Espinoza

Alberti³

et al. 2013

BMC Genomics

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BackgroundMicrobes drive the biogeochemistry that fuels the planet. Microbial viruses modulate their hosts directly through mortality and horizontal gene transfer, and indirectly by re-programming host metabolisms during infection. However, our ability to study these virus-host interactions is limited by methods that are low-throughput and heavily reliant upon the subset of organisms that are in culture. One way forward are culture-independent metagenomic approaches, but these novel methods are rarely rigorously tested, especially for studies of environmental viruses, air microbiomes, extreme environment microbiology and other areas with constrained sample amounts. Here we perform replicated experiments to evaluate Roche 454, Illumina HiSeq, and Ion Torrent PGM sequencing and library preparation protocols on virus metagenomes generated from as little as 10pg of DNA.ResultsUsing %G + C content to compare metagenomes, we find that (i) metagenomes are highly replicable, (ii) some treatment effects are minimal, e.g., sequencing technology choice has 6-fold less impact than varying input DNA amount, and (iii) when restricted to a limited DNA concentration (<1μg), changing the amount of amplification produces little variation. These trends were also observed when examining the metagenomes for gene function and assembly performance, although the latter more closely aligned to sequencing effort and read length than preparation steps tested. Among Illumina library preparation options, transposon-based libraries diverged from all others and adaptor ligation was a critical step for optimizing sequencing yields.ConclusionsThese data guide researchers in generating systematic, comparative datasets to understand complex ecosystems, and suggest that neither varied amplification nor sequencing platforms will deter such efforts.

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Section: Discussionmentioning

confidence: 99%

“…Fosmids had elevated %G + C, which is ascribed to a cloning bias [26]. Among the remaining libraries, we expected a low %G + C shift due to the adaptor-mediated amplification step, commonly attributed to inhibitory effects of high %G + C DNA secondary structures, either during library amplification [30] or downstream emPCR [31].…”

Section: Discussionmentioning

confidence: 99%

Sequencing platform and library preparation choices impact viral metagenomes

Solonenko

Ignacio‐Espinoza

Alberti³

et al. 2013

BMC Genomics

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“…The functional metagenomic approach presents two different strategies for libraries generation. Primarily, large-insert libraries, constructed in cosmids or fosmids, allow for the stable recovery of large DNA fragments and sequence homology screening purposes [8]. This strategy would also allow the recovery of complete biosynthetic pathways or the functional expression of large multi-enzyme assemblies (as in the case of polyketide synthases or hydrogenases clusters) [9,10].…”

Section: Introductionmentioning

confidence: 99%

“…On the other hand, small-insert expression libraries (i.e. lambda phage vectors and plasmids), are constructed for activity screening from single genes or small operons [8]. In this strategy, strong vector expression signals (e.g.…”

Section: Introductionmentioning

confidence: 99%

Big trouble with little inserts: boundaries in metagenomic screenings usinglacZα based vectors

LdF

et al. 2017

Preprint

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21The vast biochemical repertoire found in microbial communities from a wide-range of environments allows screening 22 and isolation of novel enzymes with improved catalytic features. In this sense, metagenomics approaches have been 23 of high relevance for providing enzymes used in diverse industrial applications. For instance, glycosyl hydrolases, 24 which catalyze the hydrolysis of carbohydrates to sugars, are essential for bioethanol production from renewable 25resources. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities 26 from microbial communities inhabiting a soil sample by using the lacZα-based plasmid pSEVA232 in the generation 27 of a screenable metagenomic library. For this, we used a functional screen based on skimmed milk agar and a pH 28 indicator dye as previously reported in literature. Although we effectively identified nine positive clones in the 29 screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded 30 in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within 31 the coding region of the lacZα gene present in the original vector. We concluded that the current method has a higher 32 tendency for false positive clones' recovery, when used in combination with a lacZα-based vector. Finally, we discuss 33 the molecular explanation for positive phenotype recovering and highlight the importance of reporting boundaries in 34 metagenomic screenings methodologies. 35

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“…The general assumption in cloning-based metagenomic approaches is that foreign DNA can be stably maintained in E. coli and that the cloned DNA is a fair representation of the original sample. However, it has been previously observed that fosmid libraries exhibit a GC bias [1, 2]. In general, such cloning biases may affect conclusions derived from analysis of the clone libraries.…”

Section: Introductionmentioning

confidence: 99%

Strong spurious transcription likely a cause of DNA insert bias in typical metagenomic clone libraries

Lam

Charles

2015

Preprint

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Background Clone libraries provide researchers with a powerful resource with which to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, as well as allowed the mining of novel enzymes for specific functions of interest. These libraries are often constructed by cloning large-inserts (∼30 kb) into a cosmid or fosmid vector. Recently, there have been reports of GC bias in fosmid metagenomic clone libraries, and it was speculated that the bias may be a result of fragmentation and loss of AT-rich sequences during the cloning process. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role in library bias. Results To explore the possible mechanisms responsible for sequence bias in clone libraries, and in particular whether fragmentation is involved, we constructed a cosmid clone library from a human microbiome sample, and sequenced DNA from three different steps of the library construction process: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final constructed cosmid library, and we provide strong evidence that the sequence bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after the DNA is introduced into E. coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for consensus promoters and found that rpoD/σ70 promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the reference genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found that the bias appears to be more closely correlated with the number of rpoD/σ70 consensus sequences in the genome than with simple GC content. Conclusions The GC bias of metagenomic clone libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter consensus sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/σ70 consensus-like in E. coli may lead to plasmid instability or loss of insert DNA. Our results suggest that despite widespread use of E. coli to propagate foreign DNA, the effects of in vivo transcriptional activity may be under-appreciated. Further work is required to tease apart the effects of transcription from those of gene product toxicity.

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Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis

Cited by 22 publications

References 43 publications

Sequencing platform and library preparation choices impact viral metagenomes

Sequencing platform and library preparation choices impact viral metagenomes

Big trouble with little inserts: boundaries in metagenomic screenings usinglacZα based vectors

Strong spurious transcription likely a cause of DNA insert bias in typical metagenomic clone libraries

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