Hi everyone,
I am looking to carry out a metagenomics project on the MiSeq or the HiSeq. I've done some reading around the subject and I found a paper by Maughan et al. that describes a protocol that uses 2 primer pairs introduced at different time points during the sequencing of the V567 region on the GA-IIx. When the second primer pair is introduced the first primer pair is removed by denaturation. The sequencing steps basically go like so:
1. Illumina primer used to sequence barcode - 8 cycles. Removed by denaturation.
2. Custom primers introduced. Forward primer anneals upstream of V5 and reverse primer anneals downstream of V7. After 36 cycles these are denatured.
3. New set of custom primers introduced. Forward binds upstream of V6 and reverse anneals downstream of V6. After 36 cycles they are denatured.
4. Fragment flips and is regenerated then the process is repeated one more time.
I was interested to know whether or not this protocol would work on the HiSeq or MiSeq?
I know there are reservoirs where custom primers can be added on the Illumina cartridge and I thought these could be used in addition to the other primer reservoirs to carry out this protocol. The sticking point for me is how the primers could be removed by denaturation?
Thanks for reading/replying.
Stan
I am looking to carry out a metagenomics project on the MiSeq or the HiSeq. I've done some reading around the subject and I found a paper by Maughan et al. that describes a protocol that uses 2 primer pairs introduced at different time points during the sequencing of the V567 region on the GA-IIx. When the second primer pair is introduced the first primer pair is removed by denaturation. The sequencing steps basically go like so:
1. Illumina primer used to sequence barcode - 8 cycles. Removed by denaturation.
2. Custom primers introduced. Forward primer anneals upstream of V5 and reverse primer anneals downstream of V7. After 36 cycles these are denatured.
3. New set of custom primers introduced. Forward binds upstream of V6 and reverse anneals downstream of V6. After 36 cycles they are denatured.
4. Fragment flips and is regenerated then the process is repeated one more time.
I was interested to know whether or not this protocol would work on the HiSeq or MiSeq?
I know there are reservoirs where custom primers can be added on the Illumina cartridge and I thought these could be used in addition to the other primer reservoirs to carry out this protocol. The sticking point for me is how the primers could be removed by denaturation?
Thanks for reading/replying.
Stan
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