"Things to Consider For Metagenomics Projects"
Things to Consider For Metagenomics Projects What is your big question? What environmental sample(s) will you be analyzing? What variables are important between your samples? What genetic information are you actually going to sample? 16S rRNA or another phylogenetically useful “universal” marker Inserts a PCR step into your protocol Primer choice now very important Functional units (specific enzyme activities) Inserts cloning and functional complementation steps into your protocol Complementation assay choice now very important Will you rely on native promoter activity or not - important to consider All genetic information Makes the fewest assumptions, but results in the most complex output Obtaining your metagenomics samples: How will you avoid contamination? How much sample is enough? DNA isolation method? ( some samples may require special processing) Will you need to amplify the entire sample prior to sequencing? Choice of sequencing method: Parameters that matter Cost Source DNA preparation required Amount of source DNA required Size of output sequences Will post sequence manipulation be required? Trimming of reads Throwing out reads with too many ambiguities Assembly into contigs? Choice of sequence analysis methods: Phylogenetic sampling of 16S or other markers You will want to bin sequences based on % identity, with user control You will want to be able to identify bins by comparison to reference genes You will want to be able to generate graphs of rank abundance, etc. True metagenomics sequencing (sequence everything) You will want to be able to compare sequences within & beyond your sample (e.g., BLAST) You will want to be able to bin sequences based on % identity Resources: Andersson et al., 2008. PLoS ONE 3:e2836. Comparative Analysis of Human Gut Microbiota by Barcoded Pyrosequencing. Dethlefsen et al., 2008. PLoS Biology 6:e280. The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing. Huber et al., 2009. Environmental Microbiology 11:1292-302. Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure. Jurkowski et al., 2007. CBE-Life Sciences Education 6:260-5. Metagenomics: A Call for Bringing a New Science into the Classroom (While It’s Still New). Krause et al., 2008. Nucleic Acids Research 36:2230-9. Phylogenetic classification of short environmental DNA fragments. Kunin et al., 2008. Microbiology And Molecular Biology Reviews 72:557-8. A Bioinformatician’s Guide to Metagenomics. Kysela et al., 2005. Environmental Microbiology 7:356-64. Serial analysis of V6 ribosomal sequence tags (SARST-V6): a method for efficient, high-throughput analysis of microbial community composition. Lazarevic et al., 2009. Journal of Microbiology Methods 79:266-71. Metagenomic study of the oral microbiota by Illumina high-throughput sequencing. Quince et al., 2009. Nature Methods 6:639-41. Accurate determination of microbial diversity from 454 pyrosequencing data. Raes et al., 2007. Current Opinion in Microbiology 10:490-8. Get the most out of your metagenome: computational analysis of environmental sequence data. Vieites et al., 2009. FEMS Microbiol Rev 33 (2009) 236-55. Metagenomics approaches in systems microbiology. Von Mering et al., 2007. Science 315:1126-1130. Quantitative phylogenetic assessment of microbial communities in diverse environments. Weizhong et al. CD-HIT Suite: Biological Sequence Clustering and Comparison (Web- accessible sequence analysis tools) weizhong-lab.ucsd.edu/cdhit_suite/cgi-bin/index.cgi Wommack et al., 2008. Applied & Environmental Microbiology 74:1453-63. Metagenomics: read length matters. Ribosomal Database Project. rdp.cme.msu.edu/