Tandem dual index- low Q30 for index 2

juliar · 02-18-2019, 02:41 AM

Hello,

While reading up about index hopping, I came across the Illumina whitepaper (https://www.illumina.com/content/dam...inkId=36607862) which states that unique dual indexes help to mitigate this.

While reading another paper about index hopping (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5759201/), where the authors used dual-matched indexes, they mention an online post by Illumina (https://support.illumina.com/bulleti...x-designs.html) which says that distinct dual indexes work better than tandem dual indexes. Illumina present data showing how tandem dual indexes have a low Q30 for the index 2 read in comparison with distinct dual indexes and data also which shows a ‘T overcall / A undercall’ phenotype for the tandem design. However, the authors of the dual-matched index paper did not find the reduced Q30 scores reported by Illumina.

So my question is why would using a tandem index affect the Q30 score for the second index read? I don't understand how data from the first index read would affect the data generated during the second index read?

Thanks in advance for any insight!

luc · 02-18-2019, 09:25 AM

The results do vary by sequencer (the Hiseq 4000 is not - or only minimally - bothered by dual matched indices); the NovaSeq OTOH can tank with these. Likely library insert sizes can also play a role.
The MiSeq and NextSeq also have no troubles with dual matched indices.

The clustering chemistry seems to be different on the NovaSeq but I have no idea what causes the problems.

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02-18-2019, 02:41 AM	#1
juliar Junior Member Location: Northern Ireland Join Date: Feb 2019 Posts: 1	Tandem dual index- low Q30 for index 2 Hello, While reading up about index hopping, I came across the Illumina whitepaper (https://www.illumina.com/content/dam...inkId=36607862) which states that unique dual indexes help to mitigate this. While reading another paper about index hopping (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5759201/), where the authors used dual-matched indexes, they mention an online post by Illumina (https://support.illumina.com/bulleti...x-designs.html) which says that distinct dual indexes work better than tandem dual indexes. Illumina present data showing how tandem dual indexes have a low Q30 for the index 2 read in comparison with distinct dual indexes and data also which shows a ‘T overcall / A undercall’ phenotype for the tandem design. However, the authors of the dual-matched index paper did not find the reduced Q30 scores reported by Illumina. So my question is why would using a tandem index affect the Q30 score for the second index read? I don't understand how data from the first index read would affect the data generated during the second index read? Thanks in advance for any insight!

02-18-2019, 09:25 AM	#2
luc Senior Member Location: US Join Date: Dec 2010 Posts: 452	The results do vary by sequencer (the Hiseq 4000 is not - or only minimally - bothered by dual matched indices); the NovaSeq OTOH can tank with these. Likely library insert sizes can also play a role. The MiSeq and NextSeq also have no troubles with dual matched indices. The clustering chemistry seems to be different on the NovaSeq but I have no idea what causes the problems. Last edited by luc; 02-18-2019 at 09:28 AM.