Data handling strategies for high throughput pyrosequencers.
BACKGROUND
New high throughput pyrosequencers such as the 454 Life Sciences GS 20 are capable of massively parallelizing DNA sequencing providing an unprecedented rate of output data as well as potentially reducing costs. However, these new pyrosequencers bear a different error profile and provide shorter reads than those of a more traditional Sanger sequencer. These facts pose new challenges regarding how the data are handled and analyzed, in addition, the steep increase in the sequencers throughput calls for much computation power at a low cost.


RESULTS
To address these challenges, we created an automated multi-step computation pipeline integrated with a database storage system. This allowed us to store, handle, index and search (1) the output data from the GS20 sequencer (2) analysis projects, possibly multiple on every dataset (3) final results of analysis computations (4) intermediate results of computations (these allow hand-made comparisons and hence further searches by the biologists). Repeatability of computations was also a requirement. In order to access the needed computation power, we ported the pipeline to the European Grid: a large community of clusters, load balanced as a whole. In order to better achieve this Grid port we created Vnas: an innovative Grid job submission, virtual sandbox manager and job callback framework. After some runs of the pipeline aimed at tuning the parameters and thresholds for optimal results, we successfully analyzed 273 sequenced amplicons from a cancerous human sample and correctly found punctual mutations confirmed by either Sanger resequencing or NCBI dbSNP. The sequencing was performed with our 454 Life Sciences GS 20 pyrosequencer.


CONCLUSIONS
We handled the steep increase in throughput from the new pyrosequencer by building an automated computation pipeline associated with database storage, and by leveraging the computing power of the European Grid. The Grid platform offers a very cost effective choice for uneven workloads, typical in many scientific research fields, provided its peculiarities can be accepted (these are discussed). The mentioned infrastructure was used to analyze human amplicons for mutations. More analyses will be performed in the future.
Trombetti
Gabriele A
GA
Institute for Biomedical Technologies-National Research Council, via Fratelli Cervi 93, 20090 Segrate, MI, Italy. gabriele.trombetti@itb.cnr.it
Bonnal
Raoul J P
RJ
Rizzi
Ermanno
E
De Bellis
Gianluca
G
Milanesi
Luciano
L
D000465:Algorithms; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D003628:Database Management Systems; D030541:Databases, Genetic; D016247:Information Storage and Retrieval / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D004247:DNA