Abstract
The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary ‘next-generation’ DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Change history
16 October 2012
This article has been corrected since Advance Online Publication and a corrigendum is also printed in this issue.
References
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W et al. (1997). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.
Béjà O, Aravind L, Koonin EV, Suzuki MT, Hadd A, Nguyen LP et al. (2000a). Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science 289: 1902–1906.
Béjà O, Suzuki MT, Koonin EV, Aravind L, Hadd A, Nguyen LP et al. (2000b). Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage. Environ Microbiol 2: 516–529.
DeLong EF . (2005). Microbial community genomics in the ocean. Nat Rev Microbiol 3: 459–469.
DeLong EF, Preston CM, Mincer T, Rich V, Hallam SJ, Frigaard N et al. (2006). Community genomics among stratified microbial assemblages in the ocean’s interior. Science 311: 496–503.
Edwards R, Rodriguez-Brito B, Wegley L, Haynes M, Breitbart M, Peterson D et al. (2006). Using pyrosequencing to shed light on deep mine microbial ecology. BMC Genomics 7: 57.
Feingersch R, Béjà O . (2009). Bias in assessments of marine SAR11 biodiversity in environmental fosmid and BAC libraries? ISME J 3: 1117–1119.
Frias-Lopez J, Shi Y, Tyson GW, Coleman ML, Schuster SC, Chisholm SW et al. (2008). Microbial community gene expression in ocean surface waters. Proc Natl Acad Sci USA 105: 3805–3810.
Ghai R, Martin-Cuadrado AB, Molto AG, Heredia IG, Cabrera R, Martin J, Verdú M, Deschamps P, Moreira D, López-García P, Mira A, Rodriguez-Valera F . (2010). Metagenome of the Mediterranean deep chlorophyll maximum studied by direct and fosmid library 454 pyrosequencing. ISME J 4: 1154–1166.
Giovannoni SJ, Britschgi TB, Moyer CL, Field KG . (1990). Genetic diversity in Sargasso Sea bacterioplankton. Nature 345: 60–63.
Hardcastle TJ . (2011). baySeq: empirical Bayesian analysis of patterns of differential expression in count data. R package version 1.6.0.
Hardcastle TJ, Kelly KA . (2010). baySeq: empirical Bayesian methods for identifying differential expression in sequence count data. BMC Bioinformatics 11: 422.
He X, Ou H, Yu Q, Zhou X, Wu J, Liang J et al. (2007). Analysis of a genomic island housing genes for DNA S-modification system in Streptomyces lividans 66 and its counterparts in other distantly related bacteria. Mol Microbiol 65: 1034–1048.
Huson DH, Auch AF, Qi J, Schuster SC . (2007). MEGAN analysis of metagenomic data. Genome Res 17: 377–386.
Kanehisa M . (2006). From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34: D354–D357.
Kanehisa M, Goto S . (2000). KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 28: 27–30.
Karl DM, Lukas R . (1996). The Hawaii Ocean Time-series (HOT) program: Background, rationale and field implementation. Deep-Sea Res Pt II 43: 129–156.
MacLean D, Jones JDG, Studholme DJ . (2009). Application of ‘next-generation’ sequencing technologies to microbial genetics. Nat Rev Microbiol 7: 287–296.
Martinez A, Bradley AS, Waldbauer JR, Summons RE, DeLong EF . (2007). Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host. Proc Natl Acad Sci USA 104: 5590–5595.
Martinez A, Tyson GW, DeLong EF . (2010). Widespread known and novel phosphonate utilization pathways in marine bacteria revealed by functional screening and metagenomic analyses. Environ Microbiol 12: 222–238.
Medlin L, Elwood HJ, Stickel S, Sogin ML . (1988). The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71: 491–499.
Olsen GJ, Lane DJ, Giovannoni SJ, Pace NR, Stahl DA . (1986). Microbial ecology and evolution: A ribosomal RNA approach. Annu Rev Microbiol 40: 337–365.
Pace NR . (1997). A molecular view of microbial diversity and the biosphere. Science 276: 734–740.
Pace NR . (2009). Mapping the tree of life: Progress and prospects. Microbiol Mol Biol Rev 73: 565–576.
Pace NR, Stahl DA, Olsen GJ, LD J . (1985). Analyzing natural microbial populations by rRNA sequences. ASM News 51: 4–12.
Pham VD, Konstantinidis KT, Palden T, DeLong EF . (2008). Phylogenetic analyses of ribosomal DNA-containing bacterioplankton genome fragments from a 4000 m vertical profile in the North Pacific Subtropical Gyre. Environ Microbiol 10: 2313–2330.
R Development Core Team (2009) R: A Language and Environment for Statistical Computing R Foundation for Statistical Computing: Vienna, Austria.
Rondon MR, August PR, Bettermann AD, Brady SF, Grossman TH, Liles MR et al. (2000). Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl Environ Microbiol 66: 2541–2547.
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT et al. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491.
Schmidt TM, DeLong EF, Pace NR . (1991). Analysis of a marine picoplankton community by 16 S rRNA gene cloning and sequencing. J Bacteriol 173: 4371–4378.
Shi Y, Tyson GW, DeLong EF . (2009). Metatranscriptomics reveals unique microbial small RNAs in the ocean’s water column. Nature 459: 266–269.
Shi Y, Tyson GW, Eppley JM, DeLong EF . (2010). Integrated metatranscriptomic and metagenomic analyses of stratified microbial assemblages in the open ocean. ISME J 5: 999–1013.
Sorek R, Zhu Y, Creevey CJ, Francino MP, Bork P, Rubin EM . (2007). Genome-wide experimental determination of barriers to horizontal gene transfer. Science 318: 1449–1452.
Stahl DA, Lane DJ, Olsen GJ, Pace NR . (1984). The analysis of hydrothermal vent-associated symbionts by ribosomal RNA sequences. Science 224: 409–411.
Stein JL, Marsh TL, Wu KY, Shizuya H, DeLong EF . (1996). Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon. J Bacteriol 178: 591–599.
Temperton B, Field D, Oliver A, Tiwari B, Mühling M, Joint I et al. (2009). Bias in assessments of marine microbial biodiversity in fosmid libraries as evaluated by pyrosequencing. ISME J 3: 792–796.
Tringe SG, von Mering C, Kobayashi A, Salamov AA, Chen K, Chang HW et al. (2005). Comparative metagenomics of microbial communities. Science 308: 554–557.
Tyson GW, Chapman J, Hugenholtz P, Allen EE, Ram RJ, Richardson PM et al. (2004). Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428: 37–43.
Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA et al. (2004). Environmental genome shotgun sequencing of the Sargasso Sea. Science 304: 66–74.
Wang L, Chen S, Vergin KL, Giovannoni SJ, Chan SW, DeMott MS et al. (2011). DNA phosphorothioation is widespread and quantized in bacterial genomes. Proc Natl Acad Sci USA 108: 2963–2968.
Wang L, Chen S, Xu T, Taghizadeh K, Wishnok JS, Zhou X et al. (2007). Phosphorothioation of DNA in bacteria by dnd genes. Nat Chem Biol 3: 709–710.
Woese CR . (1987). Bacterial evolution. Microbiol Rev 51: 221–271.
Woese CR . (2004). A new biology for a new century. Microbiol Mol Biol Rev 68: 173–186.
Acknowledgements
We thank David Karl and Matt Church, the captain and crew of the R/V Kilo Moana, and Hawaii Ocean Time Series personnel for facilitating work at sea and sample collection. We thank the members of the DeLong lab for many helpful discussions. This work was supported by a grant from the Gordon and Betty Moore Foundation (to EFD), by the Biological and Environmental Research program of the Office of Science in the US Department of Energy (to EFD), and by NSF Science and Technology Center Award EF0424599. This work is a contribution of the Center for Microbial Oceanography: Research and Education (C-MORE).
Author information
Authors and Affiliations
Corresponding author
Additional information
Supplementary Information accompanies the paper on The ISME Journal website
Supplementary information
Rights and permissions
About this article
Cite this article
Danhorn, T., Young, C. & DeLong, E. Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis. ISME J 6, 2056–2066 (2012). https://doi.org/10.1038/ismej.2012.35
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/ismej.2012.35
Keywords
This article is cited by
-
Ecosystem biomonitoring with eDNA: metabarcoding across the tree of life in a tropical marine environment
Scientific Reports (2017)
-
Functional metagenomic screen reveals new and diverse microbial rhodopsins
The ISME Journal (2016)
-
Strong spurious transcription likely contributes to DNA insert bias in typical metagenomic clone libraries
Microbiome (2015)
-
Sequencing platform and library preparation choices impact viral metagenomes
BMC Genomics (2013)
