I'm planning on sequencing an amplicon library on the MiSeq using a custom primer for read 1 and standard illumine primer for read 2.
Using a 300 cycle Miseq kit, read 1 length will be 20nt and read 2 length will be 250nt
The amplicon library is designed as follows
P5 - barcode/UMI sequence (20nt) - amplicon - nextera adaptor - P7
Using a custom read1 primer, the 1st 20 bases will be reading the barcode/UMI sequence, which has a good base-balanced design.
Read2 primer will read the 3'end of the amplicon which will always start with the same 15-20 bases and then have sequence diversity afterwards.
My question is will this be considered a low diversity library since read2 will always capture the same bases for the first 15-20 bases or is it fine since read1 is base-balanced and the bases in the first read are more important?
Appreciate any insight that people have!
Using a 300 cycle Miseq kit, read 1 length will be 20nt and read 2 length will be 250nt
The amplicon library is designed as follows
P5 - barcode/UMI sequence (20nt) - amplicon - nextera adaptor - P7
Using a custom read1 primer, the 1st 20 bases will be reading the barcode/UMI sequence, which has a good base-balanced design.
Read2 primer will read the 3'end of the amplicon which will always start with the same 15-20 bases and then have sequence diversity afterwards.
My question is will this be considered a low diversity library since read2 will always capture the same bases for the first 15-20 bases or is it fine since read1 is base-balanced and the bases in the first read are more important?
Appreciate any insight that people have!
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