Hi all,
I have had a good look through the forum posts and don't seem to find the same questions I have about preparing amplicons for sequencing (on a MiSeq) without using a 2-step PCR.
I need to sequence short amplicons from degraded DNA in a metabarcoding experiment. I have been advised repeatedly to avoid the 2-step PCR due to artefact and downstream issues. Therefore, I was wondering if the following approach would work or if there are any major do's and don't in relation to this:
1. amplifying using indexed (and variable N incorporated - it will be very low complexity) target sequence primers (in the presence of a blocking oligo to remove non-target amplification)
2. ligate P1/P2 and sequencing adaptors together (??)
3. Use biotinylation to remove unwanted P1/P1 fragments.
I see that without the PCR there is no enrichment step in this process but other than that (and therefore the clean-up), is there any way to avoid PCR steps in preparing amplicons for sequencing?
I have read the 16S and other metabarcoding protocols but they all seem to add adaptors to amplicons via PCR. I know that one commercial lab uses a ligation process but can't get details of it and I need to complete this in house, ideally without using any expensive kits. Does anyone know of a protocol that would be useful in this situation or if this query has been addressed elsewhere (in which case I would be most, most grateful for a link to it).
Many thanks in adavnce for any advice on this.
I have had a good look through the forum posts and don't seem to find the same questions I have about preparing amplicons for sequencing (on a MiSeq) without using a 2-step PCR.
I need to sequence short amplicons from degraded DNA in a metabarcoding experiment. I have been advised repeatedly to avoid the 2-step PCR due to artefact and downstream issues. Therefore, I was wondering if the following approach would work or if there are any major do's and don't in relation to this:
1. amplifying using indexed (and variable N incorporated - it will be very low complexity) target sequence primers (in the presence of a blocking oligo to remove non-target amplification)
2. ligate P1/P2 and sequencing adaptors together (??)
3. Use biotinylation to remove unwanted P1/P1 fragments.
I see that without the PCR there is no enrichment step in this process but other than that (and therefore the clean-up), is there any way to avoid PCR steps in preparing amplicons for sequencing?
I have read the 16S and other metabarcoding protocols but they all seem to add adaptors to amplicons via PCR. I know that one commercial lab uses a ligation process but can't get details of it and I need to complete this in house, ideally without using any expensive kits. Does anyone know of a protocol that would be useful in this situation or if this query has been addressed elsewhere (in which case I would be most, most grateful for a link to it).
Many thanks in adavnce for any advice on this.
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