Custom amplicon Libraries not Clustering
Hello,
My recent custom sequencing libraries don't produce reads despite good PhiX performance.
I am attempting to sequence a library of 420 bp amplicons consisting of a 20bp barcode followed by a constant region. I am pooling this library with 20% PhiX and spiking in 3.4 uL of 100 uM primers into the appropriate wells (R1, Index, R2 into 12,13,14). Sequencing yields high quality scores (84% Q30, 99% PF) with low clustering densities (160 K/mm2) and nearly 100% reads mapping to PhiX.
I can only guess that something is wrong with the design of these libraries or the custom primers but I can't for the life of me figure out what. Library follows the P5-R1-Insert-R2-I7-P7 structure and custom primers are 33 bp, 52% GC, Tm ~65C. I'd really appreciate if anyone had any insights with what could prevent clustering or Read 1 performance when the library structure is seemingly correct!
Here is a link to the Amplicon structure:
Thank you for your time.
-GU
My recent custom sequencing libraries don't produce reads despite good PhiX performance.
I am attempting to sequence a library of 420 bp amplicons consisting of a 20bp barcode followed by a constant region. I am pooling this library with 20% PhiX and spiking in 3.4 uL of 100 uM primers into the appropriate wells (R1, Index, R2 into 12,13,14). Sequencing yields high quality scores (84% Q30, 99% PF) with low clustering densities (160 K/mm2) and nearly 100% reads mapping to PhiX.
I can only guess that something is wrong with the design of these libraries or the custom primers but I can't for the life of me figure out what. Library follows the P5-R1-Insert-R2-I7-P7 structure and custom primers are 33 bp, 52% GC, Tm ~65C. I'd really appreciate if anyone had any insights with what could prevent clustering or Read 1 performance when the library structure is seemingly correct!
Here is a link to the Amplicon structure:
Thank you for your time.
-GU
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