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Old 02-01-2017, 03:54 PM   #1
Seq_learner
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Default Sequencing Primers contain in-line barcodes

Hi all,

I want to use dual-indexed paired-end sequencing of amplicon libraries on Miseq. My custom Read 1 and Read 2 sequencing primers have in-line barcodes for multiplexing the DNA samples. I would like to ask if I can omit the Index read step in this case?

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Old 02-01-2017, 06:50 PM   #2
nucacidhunter
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Do you mean indexed PCR primers? Sequencing primers cannot be indexed.
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Old 02-01-2017, 07:12 PM   #3
jdk787
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If you mean that your library inserts will contain inline barcodes (I assume at the beginning of your R1 and R2 read) that you will use to demultiplex, then yes you can omit the index read.
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Old 02-02-2017, 12:36 PM   #4
Seq_learner
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Thanks jdk787. And sorry for the confusion.
Yes, I mean that I want to add the barcodes adjacent to the sample DNA and read from the same sequencing primer as part of the sequence read.
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Old 02-03-2017, 01:52 AM   #5
SylvainL
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I use this technique sometimes... Do not forget to add some "N" at the beginning of your primers then, otherwise the colony detection may be inefficient (or you will have to spike PhiX at high percentage)... By experience, 5N are enough...
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Old 02-04-2017, 03:08 AM   #6
Seq_learner
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Thanks Sylvainl for your comment and advice. Yes, my primers have the addition of NNNN mixed bases, but only 4 Ns.
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Old 03-10-2017, 04:18 PM   #7
Vasco
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Hey Seq_learner

We are running all our illumina runs without the indexing reads (index length set to 0 prior to the run).

We additionally vary the length of our inline barcodes, which shifts all amplicons against each other, increasing sequencing diversity. Further, we switch the illumina tails for half the samples in the library (flow cel bind and sequencing primers) , so for half the samples sequencing starts from the reverse direction, further increasing diversity. We get away with using only 5% PhiX spike in with this strategy, and we don't use any N's.

See http://journals.plos.org/plosone/art...l.pone.0130324 for primer examples (Figure S1) and Figure S2 for diversity comparisons.

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