I am doing targeted sequencing using a <15000 kb panel, and because I consequently get rather low library conc (and try to limit the no of PCR cycles) I often use a somewhat large volumen of my library pools for the NAOH denaturation step prior flow cell loading (Nextseq 500 mid).
For rutine library pools the protocol says:
1. Dilute library pool to 2 nM in a 0.1% Tween buffer
2. Denature 5 uL hereof with 5 uL 0.2 M NaOH, neutralize with 5 uL 0.2 M Tris-HCl
3. Do this for all pools to be loaded.
4. Add to each pool HT1 loading buffer to a total volumen (for all pools) of 1 mL.
5. Load onto flowcell.
For the 15k panel:
1. Skip step 1 since the conc is below 2 nM
2. Denature 10 - 50 uL (depending on the conc of the library pool) in 0.2 M NaOH, neutralize with same amount of 0,2 M NaCl.
3-5. Same.
The thing is that it seems as if the cluster generation some times is very inefficient (measured as cluster density per library copies). I suspect that it could have something to do with the larger ratio of (Library+NaOH+Hcl) / HT1 compared to the rutine protocol. In a rutine setup for 4 library pools it would be 4 pools * 5 uL* 3 = 60 uL Library+NaOH+Hcl and 940 uL HT1. The same numbers for 4 40 uL pools in the alternative protocol is 4*40*3 = 360 uL and 640 uL HT1 buffer.
Some concerns could be :
1. A higher amount of Na Cl .
2. Lower molarity of the HT1 constituents
3. Insuffucient buffer capacity with less HT1 (pH is not optimal).
Does anyone have experience with low conc / high vol setup ?
Thanks.
Mads
For rutine library pools the protocol says:
1. Dilute library pool to 2 nM in a 0.1% Tween buffer
2. Denature 5 uL hereof with 5 uL 0.2 M NaOH, neutralize with 5 uL 0.2 M Tris-HCl
3. Do this for all pools to be loaded.
4. Add to each pool HT1 loading buffer to a total volumen (for all pools) of 1 mL.
5. Load onto flowcell.
For the 15k panel:
1. Skip step 1 since the conc is below 2 nM
2. Denature 10 - 50 uL (depending on the conc of the library pool) in 0.2 M NaOH, neutralize with same amount of 0,2 M NaCl.
3-5. Same.
The thing is that it seems as if the cluster generation some times is very inefficient (measured as cluster density per library copies). I suspect that it could have something to do with the larger ratio of (Library+NaOH+Hcl) / HT1 compared to the rutine protocol. In a rutine setup for 4 library pools it would be 4 pools * 5 uL* 3 = 60 uL Library+NaOH+Hcl and 940 uL HT1. The same numbers for 4 40 uL pools in the alternative protocol is 4*40*3 = 360 uL and 640 uL HT1 buffer.
Some concerns could be :
1. A higher amount of Na Cl .
2. Lower molarity of the HT1 constituents
3. Insuffucient buffer capacity with less HT1 (pH is not optimal).
Does anyone have experience with low conc / high vol setup ?
Thanks.
Mads
Comment