Microbial DNA sequencing

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14/06/2017

In 2010 the Oxford Genomics Centre partnered with the Modernising Medical Microbiology consortium in a bid to translate advances in sequencing technologies into diagnostic tools for tracking the spread of hospital infections. A major focus was to develop a robust and cost effective solution to prepare and sequence high numbers of bacterial DNA. As a result of this collaboration we are now able to offer a library preparation service ideally suited to high volumes of low complexity samples.

The majority of whole genome sequencing performed at the OGC is for microbial samples. When sequencing bacterial DNA, the GC content can contribute to the quality of data obtained. Our workflow has been specifically selected to cope with extreme genomes, from C.difficile (29% GC). to Tuberculosis (65% GC).

The library preparation steps are based on a miniaturised version of a standard gDNA protocol; fragmentation, end repair, adapter ligation and index incorporating PCR amplification. Although depth of coverage required is dependent on the analysis, due to the small genome sizes it is often necessary to multiplex high numbers of libraries for sequencing on a single lane. We routinely use a unique dual indexing strategy (avoiding index misallocation issue), commonly pooling 192 samples to be run on a single 150 bp, paired-end lane, and currently using the HiSeq 4000 (72-93 Gb per lane).

 

If you are interested in this service, here are some points to consider:

Batch size

We use liquid handling robotics for all steps in the protocol. For this reason it is more appropriate for us to handle sample numbers based on 96 and 384-well formats. Fewer sample numbers / partial plates will be considered. Please contact us to discuss your needs.

Sample submission

Due to the high sample numbers involved it is not appropriate to submit material in tubes. All samples should be submitted using appropriate plates. Details of suitable plates can be found in our guidelines.

Normalisation

Although our protocol will accommodate some variation, it is preferable that purified gDNA is normalised to 30 ng/ul, with 60 ul provided for each sample (this allows us to repeat the prep if necessary). The lowest input mass is 100 ng, and the maximum input volume is 34 ul. Although dependent on sample quality, an increased failure rate is observed with input masses below 100 ng.

QC

Concentration of all samples will be measured but to minimise cost, the assessment of quality is limited to representative numbers within batches. Final library QC is limited to concentration to determine appropriate multiplex pooling.

Failure rate

Variations in sample material quality, elution buffers and GC content will influenced the success of the library. For this type of prep we normally allow for 10 % failure before reporting back to our customers. What we consider to be a ‘failed’ library is based on our normal expectation and experience. If less than 10% of the libraries fail QC (or following discussion with the customer), those identified as ‘failed’ will be added to the pool by default and often be represented in the data. However, the actual representation (determined through demultiplexed read counts) will depend on the quality of the library.

 

Our methods for library production have been optimized to be competitive on cost, speed and efficiency and are frequently reviewed for further improvements. If you have any questions relating to microbial genome sequencing or to discuss your next sequencing project please contact us.

Further reading:

http://modmedmicro.nsms.ox.ac.uk/april-2014/

https://twitter.com/modmedmicro

http://modmedmicro.nsms.ox.ac.uk/mmm-news-and-updates/

Author: Simon Engledow