I am running a sequential ChIP sequencing experiment with sequenced DNA containing 2 unique adapter sequences. First, Y-shaped adapters are ligated onto the DNA (this will contain a unique barcode). Following a second round of IP, the eluted DNA has a second round of adapters ligated onto it via PCR purification.
My question is what is the best way to sort these different combinations of adapters? For example, after a primary IP against 2 antibodies I add either index 1 (for Primary AB1) or index 2(for Primary AB2). I then pool these two indices and perform a secondary IP using 2 different ABs. I add index 3 (for Secondary AB1) or index 4 (for secondary AB2).
Consequently, I will end up with 4 different combinations that will be sequenced. DNA with either: index 1 and index 3 index 1 and index 4 index 2 and index 3 index 3 and index4
Is there a quick way to separate out these different indices following sequencing? Are programs available to do this?
I could not understand if the adapters and barcodes are custom-made, or if they are regular Illumina adapters (assuming Illumina sequencing). After adding these adapters / barcodes, are the samples processed with a standard Illumina library prep protocol? Will these adapters and barcodes be part of the sequenced fragment, that is, will end up inside the reads?
Regular Illumina barcodes are sequenced separately from the reads and they are automatically processed by bcl2fastq / RTA software. They do not usually end up inside the reads, except for short insert fragments, but then they end up on the opposite (3') side.
In case the adapters / barcodes are custom and are sequenced in-line together the insert fragment, there is a demultiplex protocol using PhyloSift, and you could check this thread which mentions CASAVA could do this, maybe bcl2fastq retained this option.
Apologies, I should have mentioned that they are custom oligos.