Dear All,
the problem i am facing regarding is WGS. I performed toxin gene PCR and 16S specie specific primers for my bacteria and sequencing result was satisfactory . but on the result of WGS the picture was totally changed and i got different result . so need your expert opinions to fix this anguish will be thankful.
Is toxin PCR and 16S sufficient or reliable enough ?
PCR amplicon sequencing and WGS differ in a couple of points. Amplicon sequencing uses highly enriched target DNA for sequencing. For whole genome sequencing you need a pretty good DNA quality after extraction. The quality of your WGS results depends, among others, from the chemistry used for the sequencing reaction (which includes the length of the sequences) and the coverage rate over the genome. If your coverage is low, you might miss genes or of parts of the genome. Be aware that especially 16S rDNA PCR/sequencing is highly prone to contamination. Both techniques need pure cultures (colonies of pure cultures) without contamination with other bacteria.
With regard to the second question: reliable enough for what? If you want to know the ID of the bacterium in question or if you want to check for the presence of a toxin gene, than PCR amplicon sequencing is fine. WGS is more for doing genome comparisons, e.g. in molecular epidemiology.
hanks for your prompt reply , about reliable i means that if sequencing of 16s and PCR is same as required and DNA quality is also good so if after performing these test can i send sample to company for WGS ?? OR any caution for sample preparation for WGS.. PLZ guide me@Oliver Nolte.
Dear Muhammad Umar Zafar Khan
For WGS, purity of DNA is very important . The A260/A280 ratio should be >1.7 .
The required concentration of DNA depends on type (chemistry) for library preparation. E.g. for Nextera XT, 0.2ng/㎕ is OK.
DNA should not be also fragmented. You can check it using agarose gel-electrophoresis.
You cannot be sure without checking purity of DNA that WGS data will be OK only because of sufficient Sanger sequencing results of short DNA fragment as was explained by@Oliver Note
Good luck,
Marcela
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