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Old 10-29-2015, 09:09 AM   #1
incognitos
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Default Sequencing using custom primer on MiSeq - low diversity?

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!
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Old 10-29-2015, 04:48 PM   #2
nucacidhunter
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Purity of signals from initial 25 cycles is used for calculation of filtering stats to pass a cluster so you would not be able to do 20 cycles for R1. Read 2 most likely will fail due to low diversity and even the whole sequencing run might stop.
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Old 10-29-2015, 08:28 PM   #3
Brian Bushnell
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You need to use staggered-length inline adapters (this means adapters where part of them, of variable length, is sequenced with the read), or else multiplex your run with something diverse (e.g. PhiX, as a default).
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Old 10-30-2015, 03:16 AM   #4
WhatsOEver
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We do have a similar problem which we plan to resolve by using "dark" cycles. With this modification of the protocol you simply tell the sequencer to incorporate for example 15nt without "considering" them. This has the advantage that the cluster identification considers nucleotides 15-40 instead of 0-25. The disadvantage is that you will loose the first 15nt.
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Old 11-20-2015, 10:13 AM   #5
niroshan
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Illumina uses two lasers (green: G/T and red: A/C) to sequence. It is important to maintain color balance for each base of the read being sequenced, otherwise you'll run in difficulties for accurate base calling and run will most likely fail after the first few cycles.
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Old 11-23-2015, 06:52 AM   #6
pmiguel
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Quote:
Originally Posted by niroshan View Post
Illumina uses two lasers (green: G/T and red: A/C) to sequence. It is important to maintain color balance for each base of the read being sequenced, otherwise you'll run in difficulties for accurate base calling and run will most likely fail after the first few cycles.
Not true. At least not on a MiSeq running a modern version of MCS.

You do need some signal in each channel. But spiking in 5-10% phiX is sufficient to ensure that -- even if the rest of your libraries are single-plex.

--
Phillip
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Old 11-23-2015, 02:03 PM   #7
nucacidhunter
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Quote:
Originally Posted by niroshan View Post
Illumina uses two lasers (green: G/T and red: A/C) to sequence. It is important to maintain color balance for each base of the read being sequenced, otherwise you'll run in difficulties for accurate base calling and run will most likely fail after the first few cycles.
It is not true for NextSeq as well. There is no fluorophore attached to G nucleotides in two-channel SBS chemistry and A has two fluorophore. This results in emission from excitation by both lasers for A and no emission from G.
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