Hello every one,
Can you please suggest me which method should I use for sequencing a 300bp PCR products either Illumina or Oxford Nanopore. I have around 100 PCR products which are about 300bp long.
Dear Sushil Kumar Middha
I see a classic Sanger sequencing as a cheapest and sufficient solution for your project.
Regards,
Marcela
The cost of Sanger sequencing can be as low as $3/sample/reaction. With your product being so short, you may be able to get away from sequencing one strand. so you can have all of the 100 products sequenced for a few hundred bucks. However, if you need a full length coverage at a high accuracy, including the ends, you may need to sequence both stands, which means the cost will double. In this case, if you want to get away from sequencing just one strand, you may be able to design one primer a bit away from the region of your interest, and you use this primer for your sequencing, such that the high quality sequencing can start before the start of your target region. Hope this helps.
Thank you every one for your suggestions however these amplicons I would like to sequence to identify the type of bacterial species that cause infections in human being and these products were isolated from humans suffering from various bacterial infections
Sushil: Are you saying that you are working with some sort of metagenomic data, e.g., your PCR products are from conserved genes, such ribosome genes? If this is the case, NGS might be a better option for you than Sanger Sequencing. I wonder in this case if you also care about the differences amongst the 100 samples. If so, you'd have to add barcoding to the samples and the cost of library construction for 100 samples also adds up significantly. If the samples can be treated as one or two groups, then you may be able to get the sequencing done using any of the NGS platforms (e.g. Illumina or Ion Torrent) for $2000 (?).
Dear Sushil Kumar Middha
It would be more easy to recommend you something if you provide us more information about your experiment.
If you used 16S primers and the DNA was extracted from primarily sterile materials (cerebrospinal fluid, blood) I still recommend you Sanger sequencing because of probable monomicrobial infection (one sequence per patient).
In the case of polymicrobial results (e.g. DNA from sputum), WGS (Illumina in your case) is the better solution.
Regards,
Marcela
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