When analysing the read-to-contig distribution on assembled metagenomic data originated by 454 pyrosequencing, it raised the question if all regions on a prokaryotic genome have the same probability of being sequenced by this methodology.
Might the areas of accumulation of reads in a contig be produced by accumulation of reads belonging to several closely related organisms (chimeric), or contrary to "easier to sequence" regions?
Do you know any study where that question was addressed?
GC% above 70% are problematic. We produced full bacterial genome at 68% without problems. But some people have experienced problems above 70%. The problem came from the emulsion PCR step, as you might need special Taq for high GC. So I would guess all regions of a genome do not have the same probability of being sequenced with 454. But it depends also of the variability of GC% in the organsims under study. If it is metagenomic data then for sure there is no equal probabilties...
As far as I know (I work in sequencing insect symbionts by metagenomic approach), 454 have problems with GC and AT rich regions.
GC% above 70% are problematic. We produced full bacterial genome at 68% without problems. But some people have experienced problems above 70%. The problem came from the emulsion PCR step, as you might need special Taq for high GC. So I would guess all regions of a genome do not have the same probability of being sequenced with 454. But it depends also of the variability of GC% in the organsims under study. If it is metagenomic data then for sure there is no equal probabilties...
I would not worry about that. There are some biases but everything in significant amounts would be covered. The real problem of 454 is the homopolymeric regions that regardless of coverage with remain rather uncertain because is an intrinsic limitation of the method. A combo of 454 and Illumina fixes the problem
We have sequenced four Saccharomyces cerevisiae genomes using FLX-titanium sequencing chemistry and assembled them with a de-novo strategy using Newbler software. We found very low coverage in regions containing microsatellites and in omopolimeric regions. We think this low coverage was determined by the low quality of the sequences in these regions that were discarded during assembly. May be this problem is of secondary importance in procariotic genomes. I also know there are some problemes if you amplify your DNA using TempliPhy.
To answer ur 1st question,
In theoretical yes. However, in realistic that the DNA polymerase might be pre early terminate, half way, BIAS in shearing, BIAS in the machine. A wandering question, if a bacteria 5Mbp and one 10Mbps was culture can shear the DNA down to 20kbp, doubt 5Mbp might shear to lower ....
May I know the library size of this sequencing ?
Chimera read can be caused by the BIAS during library circulation for ROche Mate Pair protocol as well. Yet, it still possible give chimera reads from closely related species due to high similarity. If you are culturing prokaryotic cell, why dnt sequence by pure culture to make work easy??
To resolve microsatellite, can try Pacbio but need 454 or Illumina to correct the reads. Can try to combine 3 platform to fully resolve the genome ^.^
Besides all the technical biases already highlighted, theory predicts that the closest you are to the origin of replication the higher the probability of "being sequenced by 454 sequencing". Different techniques have shown that copy-number of genes closer to the origin is higher in exponentially growing cells.The shape of this distribution would very much depend on the growth conditions and growth rate of the population at the moment the DNA was harvested. I am not aware if this has ever been reported, though. Might be interesting to look into it...
The polymerase used can have a substantial effect on the coverage of GC rich regions. The Sanger Institute has tested several polymerases for sequencing specific prokaryotic genomes, and they found the most significant effect was given by the use of the standard polymerase for their Illumina library preparations. They switched to the KAPA HiFi PCR to remove biases and now obtain good coverage across GC rich regions (this data was presented by Mike Quail at several recent conferences).
Is Ion torrent a better option for sequencing of GC rich bacteria? I have been planning a genome sequencing project with a GC rich bacteria. So far i have been able to sequence bacteria having around 50 to 60% GC content.
Any suggestions on this?
As the ALU of the species are specific for each organisms and the sequence based on the pcr based on its chimeric regions are alone can be sequenced by 454 pyrosequencing
Our own analysis clearly suggested a preferential bias toward average GC and low GC templates. for the same reason we include control templates in each of our sequencing runs to calculate the amount of "extra" reads generated from such templates. This could be another way to reduce the bias post sequencing.
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