Ligase for DIY GAII library preps

nickb · 09-22-2009, 08:35 AM

Hi,
I've recently switched away from Illumina supplied to DIY library preps. I've been using the NEB Quick ligation kit (M2200) to ligate on my adapters. I've recently discovered this is 2,000 units/ul. The Illumina protocol uses 1 unit/ul (5 units in total). We have seen some unexpected chimeric reads in these PE libraries, where read 1 is a different chromosome to read 2. I suspect the excess of ligase maybe the reason. Although others I've spoken to are using this 2000 x ligase and seem to find it ok. What liagases are other people using? Has anyone had problems with inserts ligating to each other rather than the adaptors?
Cheers, Nick

elaney_k · 09-28-2009, 09:02 AM

Hi Nick,

AFAIK the Illumina protocol does actually use 5ul of the quick ligase from NEB (M2200) which is 2000U/ul, where did you get info to say it was 1 U/ul?

nickb · 09-29-2009, 02:26 AM

Hi Elaney,

I have an early version of the RNAseq protocol (from before illumina sold kits for mRNA libraries) this states 1 unit / ul of DNA ligase.
I've had a look at some patents - one version I found here: http://www.faqs.org/patents/app/20090088327

The ligation step is here:
Step 5) Ligation
[0270]Materials: [0271]A-tailed genomic DNA (from Step 3) [0272]Quick ligase buffer (2×) (NEB, B2200S) [0273]Annealed adaptor (15 μM) (from 4.) [0274]Quick Ligase (1 U/μl) (NEB, M2200L) [0275]PCR purification kit columns (Qiagen, 28104)

I think Illumina has just made a mistake with the Ligase concentrations on protocols and patents, as the Quick Ligase from NEB is 2000 U/ul.

Cheers, Nick

elaney_k · 09-29-2009, 03:48 AM

Ah! I had the same protocol and questioned Illumina tech support about it but never got a proper answer as it was an unsupported protocol at the time, I couldn't find any ligase from NEB to match the 1U/ul concentration so assumed it was a misprint.

BearClaw · 03-05-2010, 11:36 AM

I'm sure this has been mentioned somewhere else at seqanswers, but I thought it was relavant to this thread.....NEB has a kit containing all the reagents for sample preparation except for adaptors and primers. Catalogue number E6000. I'm thinking that this is quite a bit cheaper than the Illumina kit, even if you have to order the HPLC-purified oligos and adaptors (with the phosphorothioate or phosphate additions where needed) from your favorite oligo supplier.

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09-22-2009, 08:35 AM	#1
nickb Junior Member Location: London Join Date: Sep 2009 Posts: 2	Ligase for DIY GAII library preps Hi, I've recently switched away from Illumina supplied to DIY library preps. I've been using the NEB Quick ligation kit (M2200) to ligate on my adapters. I've recently discovered this is 2,000 units/ul. The Illumina protocol uses 1 unit/ul (5 units in total). We have seen some unexpected chimeric reads in these PE libraries, where read 1 is a different chromosome to read 2. I suspect the excess of ligase maybe the reason. Although others I've spoken to are using this 2000 x ligase and seem to find it ok. What liagases are other people using? Has anyone had problems with inserts ligating to each other rather than the adaptors? Cheers, Nick

09-28-2009, 09:02 AM	#2
elaney_k Member Location: Dublin, Ireland Join Date: Mar 2008 Posts: 55	Hi Nick, AFAIK the Illumina protocol does actually use 5ul of the quick ligase from NEB (M2200) which is 2000U/ul, where did you get info to say it was 1 U/ul?

09-29-2009, 02:26 AM	#3
nickb Junior Member Location: London Join Date: Sep 2009 Posts: 2	Hi Elaney, I have an early version of the RNAseq protocol (from before illumina sold kits for mRNA libraries) this states 1 unit / ul of DNA ligase. I've had a look at some patents - one version I found here: http://www.faqs.org/patents/app/20090088327 The ligation step is here: Step 5) Ligation [0270]Materials: [0271]A-tailed genomic DNA (from Step 3) [0272]Quick ligase buffer (2×) (NEB, B2200S) [0273]Annealed adaptor (15 μM) (from 4.) [0274]Quick Ligase (1 U/μl) (NEB, M2200L) [0275]PCR purification kit columns (Qiagen, 28104) I think Illumina has just made a mistake with the Ligase concentrations on protocols and patents, as the Quick Ligase from NEB is 2000 U/ul. Cheers, Nick

09-29-2009, 03:48 AM	#4
elaney_k Member Location: Dublin, Ireland Join Date: Mar 2008 Posts: 55	Ah! I had the same protocol and questioned Illumina tech support about it but never got a proper answer as it was an unsupported protocol at the time, I couldn't find any ligase from NEB to match the 1U/ul concentration so assumed it was a misprint.

03-05-2010, 11:36 AM	#5
BearClaw Junior Member Location: Boston Join Date: Feb 2010 Posts: 9	I'm sure this has been mentioned somewhere else at seqanswers, but I thought it was relavant to this thread.....NEB has a kit containing all the reagents for sample preparation except for adaptors and primers. Catalogue number E6000. I'm thinking that this is quite a bit cheaper than the Illumina kit, even if you have to order the HPLC-purified oligos and adaptors (with the phosphorothioate or phosphate additions where needed) from your favorite oligo supplier.