NaOH denaturation with large volumen of low conc library

madras · 09-13-2018, 10:06 AM

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

gabrieltw · 09-14-2018, 04:18 AM

Try to search older threads. There are a few talking about using low concentration library.

madras · 09-14-2018, 06:14 AM

Yeah thanks.
I did look through these, e.g. this one. They seem to be somewhat old. My main concern I think is whether HT1 buffer need to be almost "undiluted" - i.e. constitute the majority of the volumen loaded.

Kodavali · 10-22-2018, 05:21 AM

Hi,

I recently ran Nextseq, I have observed that the cluster density and the total number of reads was very low when compare to the other experiments. Qscore was 97%, Cluster passing filter was above 95% but cluster density K/mm2 was around 94 and the total number of reads for 4 lanes was around 200 million reads. For my previous experiments usually I will get above 180 K/mm2 and around 400 to 500 million reads. I have just followed the same as previous experiments, but not sure where went wrong to get low cluster density and reads.

I have denatured the sample with 0.2 N NaOH, is it the sample that is behaving or something wrong with the preparation?

I used 4nM sample conc with 1.8 loading concentration.

Thanks&Regards,
Kodavali

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09-13-2018, 10:06 AM	#1
madras Junior Member Location: Denmark Join Date: Sep 2018 Posts: 2	NaOH denaturation with large volumen of low conc library 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 4403 = 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 Last edited by madras; 09-13-2018 at 10:09 AM.

09-14-2018, 04:18 AM	#2
gabrieltw Member Location: Boston Join Date: Mar 2013 Posts: 24	Try to search older threads. There are a few talking about using low concentration library.

09-14-2018, 06:14 AM	#3
madras Junior Member Location: Denmark Join Date: Sep 2018 Posts: 2	Yeah thanks. I did look through these, e.g. this one. They seem to be somewhat old. My main concern I think is whether HT1 buffer need to be almost "undiluted" - i.e. constitute the majority of the volumen loaded.

10-22-2018, 05:21 AM	#4
Kodavali Junior Member Location: Sweden Join Date: Aug 2018 Posts: 2	Hi, I recently ran Nextseq, I have observed that the cluster density and the total number of reads was very low when compare to the other experiments. Qscore was 97%, Cluster passing filter was above 95% but cluster density K/mm2 was around 94 and the total number of reads for 4 lanes was around 200 million reads. For my previous experiments usually I will get above 180 K/mm2 and around 400 to 500 million reads. I have just followed the same as previous experiments, but not sure where went wrong to get low cluster density and reads. I have denatured the sample with 0.2 N NaOH, is it the sample that is behaving or something wrong with the preparation? I used 4nM sample conc with 1.8 loading concentration. Thanks&Regards, Kodavali