I don't know what you actually want to do (I mean genotypic wise, marker or workflow) but sanger sequencing with those many samples does not seem to be logical/practical.

Dear Abhijeet, I am planning to do genotyping on existing plasma samples ! Since we are running against time & looking at the quantum of samples, is it wise to go for NGS ! These samples have already been confirmed of their reactivity status by immunoassays (ELISA / CLIA / WB).

Are the costs of the methods also a consideration? Depending on the targets of detection, Sanger sequencing might costs more per sample, but the subsequent analyses is much easier (less costly). On the other hand, NGS (you also need clarify that which platform/method) also depending on the targets of detection (single gene or multiple genes?), the cost of library prep kits, NGS run reagents, and most importantly bioinoformatic analyses, may cost more eventually.

Have you heard of Hologic Panther system, it is automated system that can be used to detect large batches of samples for common pathogens, such as HIV, HBV, Flu etc. One of labs in our department is helping Hologic test run their reagents for detecting HIV in human plasma sample. They have tested over 4,000 samples so far, the results are pretty encouraging. :-)

Dear Kejun,

I m not bothered about the cost as of now, immediate concern is to get maximum samples genotyped...... alongside respective NIBSC standards. What's your take on template-independent next-generation sequencing (TI-NGS) assay !

I have never done TI-NGS assay before. After reading the Wei, B. at all's Journal of Molecular Diagnostics paper, it seems to me that it is really similar to RNASeq. Since you are not too concern about the cost, then the consideration should be whether you or your have someone that can perform the subsequent bioinformatic analyses to extract the sequence information you need? Because, as I mentioned before that the complexity of the analysis is much more that the regular old Sanger sequencing.

I understand that should'nt be much of a concern to do the Bioinformatics analysis post sequencing. My only concern is whether if its a wise thing to do & the turn around time vis-a-vis the number of samples....m also looking for a lab who can take up d job (outsource) & do the NGS !

For making decision about the sequencing platform, you also need to consider the read length and depth of sequencing you want to get. Its a very important criteria before you really do sequencing. As I told you before, sanget sequencing is not a good method. On the other hand I don't understand why Kejun Guo is emphasizing so much about the higher cost in NGS data analysis. For me it does not sound logical. Sanger sequence is not like perfect out of the machine and you can use them right away, they also need to be processed. Rather it can be more expensive both time and money wise to go for Sanger sequencing with thousands of sample. The main principle of NGS is to multiplex and fast analysis itself. If this could be easily done with Sanger, I don't think there would be so many methods for massive parallel sequencing.

Abhijeet Singh, If you work in a lab that is funded by grants, cost is always a concern. Sanger sequencing has higher initial investment on sample processing and preparation, but the sequence analysis is fairly simple, just basic alignment. NGS however, you can just prepare the RNA and send out. However, you do need capability to analyze the high-throughput data afterwards. For that, you can not completely rely on others to do the analyses for you, not just for concern of the cost, in my opinion. Otherwise, it will be very inconvenient when it comes to write the manuscripts, every little change you want to make, you'll have to contact the people that did the initial analyses and rerun them.

Chandranand: Theoretically, TI-NGS is definitely applicable for your experiment design. However, you do need consider the sequencing depth if you need the full length sequence of the target genes. If you just need the matched counts of the pathogen genes, that will be very straight forward.