Originally posted by nucacidhunter
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Last edited by Brian Bushnell; 10-10-2017, 01:10 AM.
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Originally posted by Markiyan View PostIn that case we need to have first 4-5 bases as a random sequence (for clusters ID), followed by the barcode (another 4-8 bases), than the insert...
This is really important for RNAseq & CHIPseq applications, where we want to keep the cross-talk bellow 10^-6...
Classical dual unique indexing would get us into 10^-4 - 10^-5 range.
BTW: Actually the spatial signal separation from the patterned flowcell clusters should resemble more the 454 signal, and the clusters should be much more easier to call, even if low diversity at the first few bases.
1- it will be difficult to obtain good oligo annealing to form adapters and also there will unpaired oligos which would require further purification
2- Illumina is unlikely to change their established indexing strategy and also there might be some patents on inline barcoding
Unique duel indexing (every library with different index 1 and 2) should enable identification and filtering of any fragments resulting from index hopping.
Actually in patterned flow cells position of each nano-well which would have clusters is already known. I have not thought about the need for sequence diversity but it could be related to background signal.
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Originally posted by nucacidhunter View PostThanks. IDT has not released their UDI adapters that they are co-developing with Illumina. NuGEN adapters are not sold as a stand alone product and I am not sure if their adapters are specific to their kits or if they can be used with other kits as well.
The UDI 384 are 10 base indexes though. So you would have to reduce your sequence insert reads by 2 bases each to compensate...
NuGEN sales reps told me that their adapters don't use T/A tailing. So they wouldn't work with most other kits.
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Phillip
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Originally posted by Brian Bushnell View PostIt only works for applications that are not sensitive to crosstalk. Personally, I would never multiplex samples of the same genus on a NovaSeq unless all libraries had dual unique barcodes.
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Phillip
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Originally posted by pmiguel View PostDid you see much phiX index hopping? Searching the "undetermined" fastq for index hops involving one of i7/i5 GGGGGGGG/TCGTAGTG I tentatively identified a fair number. Of course they may just be the result of signal bleed.
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Phillip
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Originally posted by luc View PostI am not sure that index hopping onto PhiX reads would be a good measure. As far as I remember the Illumina PhiX adapter sequences are quite different from TruSeq or Nextera sequences.
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PhillipAttached Files
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List price on S4 reagent kits is about $30K. For NovaSeq sites with ≥5 NovaSeq instruments, they're able to get a discount large enough to achieve the "20% cheaper than HiSeq X" statement Francis deSouza made when they announced this platform. Something tells me this is factoring in how many samples could be run per year on NovaSeq than HiSeq X due to the throughput and runtime, so their discount is likely not more than 20% off of list. Everyone else likely gets <10% discount. HiSeq 4000 (300 cycle) kits are about $17K list price, so they're not the same price but S4 will give you about 5x the output (in about half the time).
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