So I'm troubleshooting V2 vs. V3 clustering with the same exact samples (both of long and short inserts, all enriched gDNA libraries) and I wanted to see if anyone else has done any similar work.
See some recent data which shows generally half the cluster densities, as well as very poor performance of longer insert libraries on V3 kits.
So I went sleuthing on why this might be...suspecting changes to the clustering recipes....
Illumina calls out the only reagent differences are scan and incorporation mix, so I (*admittedly naively*) don't think they'd change the formulation of clustering reagents.
I noted what I think are significant differences in clustering recipes (by looking at the recipe .xml's for "OnBoardClusterGeneration" steps, both are attached for the Default chemistry).
1. V2 chemistry pumps the template in first, then heats up to 75C, while V3 heats the FC up to 75C, then pumps the template in. (lines ~18-20 of the attached files).
2. The template rampdown step ("TMP RampDown") to 40C is twice the time for V3 than it is for V2.
3. "First Extension" is dramatically different as well. V3 is at 50C, followed by 6 cycles of pump-wait. V2 doesn't appear to change the temperature to 50C (so it's still at 40C from the Rampdown??), and then performs 15 cycles of pump-wait (with half the volume of reagents).
4. "Amplification1" is very different as well. V2 doesn't wait at all in between each reagent, but just waits for 15000 after the AMS1 is injected. V3 pumps each reagent (LDR, LPM, AMS1) and waits in between for 7200,7200,18700 respectively. Also V2 does 26 cycles, while V3 does 24.
5. "Linearisation" is similarly lengthened in V3 as in the previous step, adding waits in between reagent pumps (and cleaving for roughly 3x the time).
So I'm talking to Illumina TS, but I'm going to try running the V2 clustering recipe with a V3 kit, some of these mods seem significant that they could impact general clustering efficiency as well as long insert amplification. Any input would be appreciated, even if it's an Illumina employee telling me to stop reading recipe .xmls.
See some recent data which shows generally half the cluster densities, as well as very poor performance of longer insert libraries on V3 kits.
So I went sleuthing on why this might be...suspecting changes to the clustering recipes....
Illumina calls out the only reagent differences are scan and incorporation mix, so I (*admittedly naively*) don't think they'd change the formulation of clustering reagents.
Yes. There are two new reagents in MiSeq v3 kits:
The PR2 volume is increased to 500 ml to support longer runs. In the near future, all MiSeq PR2 bottles will have a 500 ml fill to avoid confusion when starting a run. Look for announcements on the Illumina Tech Support Bulletin Board regarding this change.
- New incorporation mix (IMT)
- New scan mix (USM)
The PR2 volume is increased to 500 ml to support longer runs. In the near future, all MiSeq PR2 bottles will have a 500 ml fill to avoid confusion when starting a run. Look for announcements on the Illumina Tech Support Bulletin Board regarding this change.
1. V2 chemistry pumps the template in first, then heats up to 75C, while V3 heats the FC up to 75C, then pumps the template in. (lines ~18-20 of the attached files).
2. The template rampdown step ("TMP RampDown") to 40C is twice the time for V3 than it is for V2.
3. "First Extension" is dramatically different as well. V3 is at 50C, followed by 6 cycles of pump-wait. V2 doesn't appear to change the temperature to 50C (so it's still at 40C from the Rampdown??), and then performs 15 cycles of pump-wait (with half the volume of reagents).
4. "Amplification1" is very different as well. V2 doesn't wait at all in between each reagent, but just waits for 15000 after the AMS1 is injected. V3 pumps each reagent (LDR, LPM, AMS1) and waits in between for 7200,7200,18700 respectively. Also V2 does 26 cycles, while V3 does 24.
5. "Linearisation" is similarly lengthened in V3 as in the previous step, adding waits in between reagent pumps (and cleaving for roughly 3x the time).
So I'm talking to Illumina TS, but I'm going to try running the V2 clustering recipe with a V3 kit, some of these mods seem significant that they could impact general clustering efficiency as well as long insert amplification. Any input would be appreciated, even if it's an Illumina employee telling me to stop reading recipe .xmls.
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