mothur.

The past few months I have been working with a microbial ecology toolkit called mothur.  So far, it is the most flexible tool I have found to calculate distance matrices from 16S alignments and, subsequently, cluster these sequences into OTUs.  Many people have asked me about how to use mothur so this post will serve as a tutorial.

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ISME Day 4

Today was the last day of ISME and a great conclusion to the conference.  Today’s theme seemed to surround microbial methods and my favorite talks were split between 2 subterranauts, Karen Lloyd and Roland Hatzenpichler.  Karen spoke in the archaea themed session about exploring single cell genomes and exploring novel enzyme function in the uncultured MCG archaea group and  Roland presented in the single cell session about a new technique to visualize newly synthesized proteins in situ.

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ISME Day 3

Today was a busy day but, at the end of the day, the one talk that stands out in my mind is Ruth Ley’s talk on the human microbiome.  Personally, I find this topic to be extremely interesting and Ruth’s presentation did not disappoint.  The talk began with an introduction to a phenomenon in human history: the introduction of agriculture.  Now, we can all imagine that during the shift from hunting and gathering to farming the human microbiome changed to adjust to the new diet.  Perhaps one of the most important changes was the introduction of starch heavy foods that were not the basis of hunter-gather diets.  One gene in particular, AMY1, codes for  Continue reading

ISME Day 2

There were some really cool sessions at ISME today.   The morning started off with a plenary talk by Lars Peter Nielsen about cable bacteria and I sampled the sessions on “Unusual strategies of microbial energy acquisition,” “Network microbial ecology,” and “Meta-ome information to microbial ecology.”  For each session I have picked a favorite talk starting with Victoria Orphan‘s talk on the archaeal-bacterial partnerships responsible for sulfate-coupled anaerobic oxidation of methane (AOM). Continue reading

ISME Day 1

This week I am at the International Society of Microbial Ecology (ISME15) Conference in Seoul, South Korea!  I want to share with you some of my favorite talks from each day.  Today, I spent most of my time in the “Microbiomes of marine ecosystems” session and made some stops at other sequencing related talks.  Perhaps my favorite talk of the day goes to… Antje Boetius!  Her talk focused on the microbial diversity of the surface of seafloor sediment and  Continue reading

Sequencing Depth and World Cup Paninis

The World Cup is underway and I am back to the blog!  I was recently inspired by the World Cup Paninis to write a post about sequencing depth.  In sequencing, the problem we are often faced with is whether or not enough sequences have been generated to be representative of a population.  Tools we often use to determine whether we have sampled enough are rarefaction curves and the Chao estimate but how many sequences would we need to generate in order to capture a 16S from every organism present in an environment? Continue reading

MetaVelvet

In my last post, I described what a De Bruijn Graph assembler is and will now go into a short tutorial of how to begin using MetaVelvet.  MetaVelvet is an extension of the popular single genome De Bruijn Graph assembler, Velvet, and is optimized to handle the varying coverage and diversity of genomes in metagenomic samples and is executed through 3 steps: velveth, velvetg, and meta-velvetg.

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De Bruijn Graph Assembly

When our lab got its first metagenomic dataset, the first thing we did was upload our QC filtered and merged paired-end Illumina reads (mean length 160 bp) onto MG-RAST for annotation.  However, when the annotations came back, some organisms whose genomes were known to be present in both our sample and the m5nr reference dataset were missing and, for those sequences that were annotated, the designated e-values centered around 1e-10.  In order to improve the annotation of our data, we decided to perform an assembly.   Searching the literature, I found that a class of assemblers — called De Bruijn Graph assemblers — were the popular choice for assembly of short read metagenomic data; however, the intuition behind how these assemblers worked was a little less clear. Continue reading