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I have successfully used the original single-digest RADseq protocol in the past. Quickly: this protocol digests individual DNA with a single RI (commonly Sbf1). Then, P1-adaptors (each containing a unique barcode to mark individual DNA) are ligated to the sticky end left by the RI. P1-barcode ligated DNA from several individuals is then pooled per library, followed by shearing and P2-adaptor ligation. After some clean-ups and an amplification PCR of RADtags, the library can be sequenced.
In the past, I have always sequenced one RAD-library per HiSeq Illumina lane. Hence, there was no need for me to have an indexed P2-adaptor. However, I'd now like to adopt my protocol for NovaSeq, which means, I need to design several new P2-adaptors each with a unique index. Thus, each library will get its unique index, allowing me to sequence multiple libraries within the same NovaSeq flowcell.
I was recommended to use the common single-index TruSeq adaptor as a template to construct my new RADseq P2-adaptors. However, RADseq P2-adaptors have some special properties, including a divergent Y-structure. So this makes the design of new P2-adaptors harder.
Does anyone have indexed P2-RADseq adaptors they have used successfully to construct single-digest RADseq libraries? If so, would you be willing to share the sequences of these P2-adaptors with me? I'd like to have a total of 10 different P2-adaptors (each with a unique index) so that I will be able to multiplex 10 RADseq libraries and sequence them together on a single Novaseq flowcell.
Thank you a lot for your help!
In the past, I have always sequenced one RAD-library per HiSeq Illumina lane. Hence, there was no need for me to have an indexed P2-adaptor. However, I'd now like to adopt my protocol for NovaSeq, which means, I need to design several new P2-adaptors each with a unique index. Thus, each library will get its unique index, allowing me to sequence multiple libraries within the same NovaSeq flowcell.
I was recommended to use the common single-index TruSeq adaptor as a template to construct my new RADseq P2-adaptors. However, RADseq P2-adaptors have some special properties, including a divergent Y-structure. So this makes the design of new P2-adaptors harder.
Does anyone have indexed P2-RADseq adaptors they have used successfully to construct single-digest RADseq libraries? If so, would you be willing to share the sequences of these P2-adaptors with me? I'd like to have a total of 10 different P2-adaptors (each with a unique index) so that I will be able to multiplex 10 RADseq libraries and sequence them together on a single Novaseq flowcell.
Thank you a lot for your help!