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Amplicon seq and targeted seq

What is the difference between amplicon sequencing and targeted sequencing? How to decide when to use either of them?

sequencing next-gen

Hello mandar.bedse!

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Not primarily bioinformatics

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Could you let me know on why this is irrelevant for this site?

Hi mandar.bedse

This is more related to wet lab sequencing approaches/protocols rather than bioinformatics. You could find the answer if you could google well enough.

Amplicon sequencing is a type of targeted sequencing. You may enrich your data with multiplex PCR-based or hybridization-based techniques. The difference for the user is mainly which type of sequencing machines you currently have - usually people make this choice looking at what the manufacturer is recommending for this particular machine (e.g., IonTorrent machines are usually using multiplex-PCR based approaches, while people tend to use hybridization techniques with Illumina - even if you can exchange the preparation protocol, this is kinda historical thing, as far as I know).

There are various challenges in bioinformatics here too. Multiplex PCR data is usually distributed around log-normal while hybridization-based techniques can be modelled with Negative Binomial quite efficiently. Hybdridization with long probes (120 for Agilent kits) gives you more "stable" enrichment while, when you have even a short indel or mismatch in your amplicon's hybridization site (around 20 basepairs if I remember correctly) - you may have almost 0 product from this region.

I'd say we use amplicons when we need extremely high coverage (>10.000 on average) and hybridization otherwise. Another challenge for the bioinformatics software working with amplicons - deduplication process. Various barcoding techniques are used for that (it is not possible to robustly resolve this issue with bioinformaitcs only)

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