ena-STUDY-CSISP-13-06-2013-10:13:27:131-30
Direct sequencing of low DNA amount
Background. The large amount of DNA needed to prepare a library in next generation sequencing protocols impedes direct sequencing of limited DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on f29 polymerase. However, this technique can be biased by the GC content of the sample, is prone to the development of chimeras, and suffers from contamination during enrichment, which contributes to undesired noise during sequence data analysis and also mars the proper functional and/or taxonomic assignments. Hypothesis. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility to sequence the genome of Escherichia coli from the minimum number of DNA molecules required for pyrosequencing according to the notion of one-bead-one-molecule. Results. Using an optimized protocol for DS, we constructed a shotgun library containing a minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gather most of the reference genome extension with a uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications.  Conclusions. The optimized DS protocol allows to perform an unbiased sequencing from samples with a very low DNA amount.
Background. The large amount of DNA needed to prepare a library in next generation sequencing protocols impedes direct sequencing of limited DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on f29 polymerase. However, this technique can be biased by the GC content of the sample, is prone to the development of chimeras, and suffers from contamination during enrichment, which contributes to undesired noise during sequence data analysis and also mars the proper functional and/or taxonomic assignments. Hypothesis. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility to sequence the genome of Escherichia coli from the minimum number of DNA molecules required for pyrosequencing according to the notion of one-bead-one-molecule. Results. Using an optimized protocol for DS, we constructed a shotgun library containing a minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gather most of the reference genome extension with a uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications.  Conclusions. The optimized DS protocol allows to perform an unbiased sequencing from samples with a very low DNA amount.