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Hope somebody nice, and clever, out there might be able to give me advice...I am a bit clueless about this and have quite a small brain too...
I've got some samples that i ran on sureselect using the low starting amount protocol.
Clinical samples from FFPE...one patient waiting for rare tumour results fairly urgently.
It seemed to go well and the libraries looked not brilliant but okay and about the right size on the chip. The cluster and sequencing reports looked fine. Now when I took it to be demultiplexed the guy cannot get any data out (few hundred reads only).
I wondered if its because this protocol using custom home made adapters and oligos designed to go with the original sureselect kit (2010) before they introduced indexes, I used the indexing postcapture PCR primers from a more recent kit...would that have stoped barcodes being attached while still amplifying the capture product??? I did have a look through the old protocols and the names of the adapters and primers are different.
I also wondered if the degraded DNA had stopped the adapters from ligating but presumably there would be no sequence at all if so, and no amplification on the PCR steps?
I have extremely limited amounts of material but have saved some from after precapture PCR enough to do another hybridisation, and I would be very grateful for some advice about how to proceed after this.
I've got some samples that i ran on sureselect using the low starting amount protocol.
Clinical samples from FFPE...one patient waiting for rare tumour results fairly urgently.
It seemed to go well and the libraries looked not brilliant but okay and about the right size on the chip. The cluster and sequencing reports looked fine. Now when I took it to be demultiplexed the guy cannot get any data out (few hundred reads only).
I wondered if its because this protocol using custom home made adapters and oligos designed to go with the original sureselect kit (2010) before they introduced indexes, I used the indexing postcapture PCR primers from a more recent kit...would that have stoped barcodes being attached while still amplifying the capture product??? I did have a look through the old protocols and the names of the adapters and primers are different.
I also wondered if the degraded DNA had stopped the adapters from ligating but presumably there would be no sequence at all if so, and no amplification on the PCR steps?
I have extremely limited amounts of material but have saved some from after precapture PCR enough to do another hybridisation, and I would be very grateful for some advice about how to proceed after this.
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