One of my Bioinformatics students, Laura Cox, is working on a Human Microbiome Project study with Martin Blaser. Yesterday, she presented a lab report to a standing room only crowd about her sequencing work on bacterial populations using the Illumina MiSeq machine. Up till now, HMP work was about the only sequencing that we consistently ran on the 454 machine. Laura showed that with the MiSeq paired-end 150 bp sequencing protocol, it was possible to sequence 16S amplicons (in the V4 region) from both ends and stitch them together using the ea-utils FASTQ-join [Erik Aronesty (2011). ea-utils : "Command-line tools for processing biological sequencing data"; http://code.google.com/p/ea-utils] to get about 260 bp reads on each amplicon. Laura used a custom multiplex scheme to get 192 different samples into one MiSeq run, which after demultiplexing, gave about 2,000 P-E reads per sample.
She also demonstrated that the resulting sequence data could be processed with QIIME [http://www.qiime.org] to get reasonable taxonomy information, build phylogenetic trees, and apply all the cute tools to calculate diversity and compare groups of samples by PCA and UNIFRAC [http://bmf2.colorado.edu/unifrac].
The economics of MiSeq are persuasive. It is giving amplicon data at about 40X less cost than 454. As our HMP protocols shift over to MiSeq, this will be the last year that we keep the 454 machine in the Genomics Core Lab.
O-MAPping the cell for a spatial understanding of basic biology
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Mapping is crucial in understanding and contextualizing our environments.
Mapping serves many purposes – it helps us establish efficient paths
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1 day ago
7 comments:
Hi there,
I would be ecstatically happy if you could publish the primers used. Have had a poke around the internet and have not yet been able to find them.
Thank you in advance,
Chris
+1!!
Hi Chris,
The primers are the same as the ones used in the Caporaso 2012 ISME article. The supplemental material has detailed methods and full primer sequences.
http://www.nature.com/ismej/journal/vaop/ncurrent/full/ismej20128a.html
-Laura
Curious about your pooling protocol and how much PhiX to spike with to make this work. Caporaso paper pooled only 24 samples together, but 192 is much better, and 2000 reads is perfectly adequate for most applications. We are soon acquiring a MiSeq in our lab and wish to quickly establish an amplicon sequencing protocol.
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