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Dear Users
I am an absolute newbie on NGS and wish to sequence about 25 genes on the MiSeq using an amplicon based approach for library preparation. I have some doubts and i hope that someone will help
I would like to ask some questions on targeted gene sequencing which utilizes an amplicon based approach for library preparation (about 250 amplicons).
I wish to synthesize primers from IDT or similar company. Cost is an concern so I would like to use this approach.
My question pertains to designing the primer. I understand that the design of the forward and reverse primer would include the illumina sequencing adapter (p5, p7), barcode and the actual forward or reverse target primer.
I have the adapter sequences from illumina.
My questions are as follows:
1: Do the barcodes have to be present only on both the forward and reverse primers, or just the reverse is OK?
2: What is the permissible length of the insert (given that 120bp for both the adapters, 30bp for both the target sequence primers). And with that logic what is the maximum allowable length of the entire amplicon (400bp, 450bp?) for the illumina MiSeq platform (given that max read length on MiSeq is 250bp)
3:When you design these amplicons, should they overlap and if so by how many bases?
I am looking forward to your reply, hope someone does reply!
Nikhil
I am an absolute newbie on NGS and wish to sequence about 25 genes on the MiSeq using an amplicon based approach for library preparation. I have some doubts and i hope that someone will help
I would like to ask some questions on targeted gene sequencing which utilizes an amplicon based approach for library preparation (about 250 amplicons).
I wish to synthesize primers from IDT or similar company. Cost is an concern so I would like to use this approach.
My question pertains to designing the primer. I understand that the design of the forward and reverse primer would include the illumina sequencing adapter (p5, p7), barcode and the actual forward or reverse target primer.
I have the adapter sequences from illumina.
My questions are as follows:
1: Do the barcodes have to be present only on both the forward and reverse primers, or just the reverse is OK?
2: What is the permissible length of the insert (given that 120bp for both the adapters, 30bp for both the target sequence primers). And with that logic what is the maximum allowable length of the entire amplicon (400bp, 450bp?) for the illumina MiSeq platform (given that max read length on MiSeq is 250bp)
3:When you design these amplicons, should they overlap and if so by how many bases?
I am looking forward to your reply, hope someone does reply!
Nikhil