Geoff Smith from Illumina presented a great piece of work his group have been collaborating on with Jill Banfield’s research group at the University of California Berkely, Department of Earth and Planetary sciences. Geoff included a discussion on their use of Moleculo.
They have been using NGS to analyse the communities of microbes performing bioremediation in soil samples from a former uranium mill in Rifle,Colorado. These microbes were collected from a core drilled at the site and sampled at different depths. The site used to process uranium and rain carries dissolved heavy metals into the groundwater and the Colorado river. Microbes internalise the heavy metals making them insoluble and assayable in the sediment core.
Using NGS the team are investigating the diversity of the microbial community and hope to understand how bioremediation might be improved by modulating this community diversity. The bacterial community is “fed” with acetate under reducing conditions to stimulate uranium absorption.
The collaboration with Illumina involved preparation of Nextera libraries for standard Illumina sequencing to try and understand the metagenome of the community. At the same time they also prepared Moleculo libraries to see if the long reads from this technology would improve their discovery efforts.
Moleculo works by first fragmenting 1ug of genomic DNA to 10kb, adapters are ligated that allow a universal clonal lrPCR to take place in a reduced-representation fo the genome. These lrPCRs are then used as the input to a Nextera library prep, pooled and sequence. The sequences can then be demultiplexed to the Moleculo barcodes and used in a de novo assembly. Find out more about Moleculo here.
What does Moleculo bring to the table: Standard Nextera libraries showed a reasonably diverse community with many of the species expected. However then Moleculo data revealed that there were many species missed in the Nextera dataset many of the low abundance species are only found using Moleculo technology. The theory is that normal sequencing leaves lots of reads that align poorly to current genomes so appear to be insignificant. But the Moleculo technology makes sense of the data by allowing the de novo assembly of reads to build larger contigs that can be made sense of.
It will be interesting to see if the same increase in species abundance is seen in other metagenomic datasets using Moleculo. It’s not just cancer that benefits from long reads after all!