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Hello everybody. I could use some help with planning for a sequencing run I'm about to start.
I will be sequencing a library where the read1 sequence is expected to be highly diverse during the first ten bases, followed by literally the same sequence across the entire library. All sequencing primers are custom. We have sequenced similar libraries in the past with these primers successfully, but in combination with other libraries with diversity across a longer sequence stretch. I'm using a NextSeq 500/550 with a High Output Kit v2.5 (75 Cycles), and I would also read index 1, index 2, and read 2 sequences which are highly diverse.
Because I don't really care about the read1 sequence after the variable region, I originally planned to use only 10 cycles for read 1 to avoid diversity issues, but I see it recommended to sequence for 26 bases for accurate quality metric calculations. What's exactly the failure mode if I sequence into the constant region? Would reads drop out of the final output? Or would I just get sequences with good quality up to base 10 but really bad quality at base 11 and beyond? The first would be disastrous but the second is perfectly acceptable.
What is the failure mode if I just run read1 for 10 cycles? I see in the NextSeq manual that template generation requires only the first 5 cycles, so if I just get somewhat inaccurate quality metrics afterwards I think that would be acceptable. If reads will drop out of the output due to underestimated quality scores, is there any way to know how many that would be?
I saw that another recommendation is to use PhiX and spike my custom primers into the standard primer wells as indicated here. What percentage would be recommended for my situation, in which the first 10 bases of read 1 are highly diverse and the rest is constant?
Thank you for your help.
I will be sequencing a library where the read1 sequence is expected to be highly diverse during the first ten bases, followed by literally the same sequence across the entire library. All sequencing primers are custom. We have sequenced similar libraries in the past with these primers successfully, but in combination with other libraries with diversity across a longer sequence stretch. I'm using a NextSeq 500/550 with a High Output Kit v2.5 (75 Cycles), and I would also read index 1, index 2, and read 2 sequences which are highly diverse.
Because I don't really care about the read1 sequence after the variable region, I originally planned to use only 10 cycles for read 1 to avoid diversity issues, but I see it recommended to sequence for 26 bases for accurate quality metric calculations. What's exactly the failure mode if I sequence into the constant region? Would reads drop out of the final output? Or would I just get sequences with good quality up to base 10 but really bad quality at base 11 and beyond? The first would be disastrous but the second is perfectly acceptable.
What is the failure mode if I just run read1 for 10 cycles? I see in the NextSeq manual that template generation requires only the first 5 cycles, so if I just get somewhat inaccurate quality metrics afterwards I think that would be acceptable. If reads will drop out of the output due to underestimated quality scores, is there any way to know how many that would be?
I saw that another recommendation is to use PhiX and spike my custom primers into the standard primer wells as indicated here. What percentage would be recommended for my situation, in which the first 10 bases of read 1 are highly diverse and the rest is constant?
Thank you for your help.