Agilent Bioanalyzer 2100 detects no DNA

obischof · 01-06-2011, 08:26 AM

Dear All,

I have run into a huge problem with our seq platform. The problem is as follows. We have prepared chromatin using Mn digestion after PFA XL. We verified the appearance of primarily mono-nucleosomes in an agarose gel. We then went ahead to chip using different abs vs histone mods. The chipped material we again analysed on a gel and we saw nice bands in the vicinity of 100-200. This material we had analysed by the Agilent Bioanalyzer but zilch. So, we ignored this in the meantime because the gel was fine. Next, we prepared the library and ran it on the Bioanalyzer. Now, we saw a nice band around 200bp-ish, which we thought was fabulous (see photo). So, we made a test run on the Illumina Sequencer and to our suprise zilch. Now, what in heavens name is happening here. If someone could help me out.

Thank you

ECO · 01-06-2011, 08:31 AM

Could your chipped material have been single stranded?

obischof · 01-06-2011, 08:50 AM

how could that be? what would that explain? Could we still build a library from that?

ECO · 01-06-2011, 12:43 PM

Well...depending on what dyes you're using on the gels where you see product, you may be seeing ssDNA that doesn't stain well or run predictably on the bioanalyzer.

And you wouldn't be able to make a good library from ssDNA either...not sure what we're looking at on the bioanalyzer.

It doesn't make sense that you got _nothing_ from the sequencing, because if you've truly amplified proper fragments you'd at least get sequence from that (maybe not biologically relevant). Was there any feedback as to what failed on the GA? No clusters? No mapped reads?

epistatic · 01-10-2011, 10:55 AM

The majority of the ChIP-DNA I get for library preps have a large quantity of ssDNA. The process of removing the cross-linking is rough and possibly denatures the sample. There are also some that think the IP also pulls down targets naturally bound to ssDNA (but I would think in the minority).

For all ChIP, the nanodrop was misleading for concentration. I now require dsDNA specific assays as initial QC/QA (picogreen, Qubit).

If there is a question of the origin of the DNA on the second bioanalyzer traces, dilute some and PCR with the Illumina primers. You should only see an increase of the material with adapters.

obischof · 01-11-2011, 08:15 AM

We had no clusters that passed the internal QC of GA, though the first cycle was OK.

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01-06-2011, 08:31 AM	#2
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	Could your chipped material have been single stranded?

01-06-2011, 08:50 AM	#3
obischof Member Location: france Join Date: Jan 2011 Posts: 11	how could that be? what would that explain? Could we still build a library from that?

01-06-2011, 12:43 PM	#4
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	Well...depending on what dyes you're using on the gels where you see product, you may be seeing ssDNA that doesn't stain well or run predictably on the bioanalyzer. And you wouldn't be able to make a good library from ssDNA either...not sure what we're looking at on the bioanalyzer. It doesn't make sense that you got _nothing_ from the sequencing, because if you've truly amplified proper fragments you'd at least get sequence from that (maybe not biologically relevant). Was there any feedback as to what failed on the GA? No clusters? No mapped reads?

01-10-2011, 10:55 AM	#5
epistatic Senior Member Location: Dronning Maud Land Join Date: Mar 2009 Posts: 129	The majority of the ChIP-DNA I get for library preps have a large quantity of ssDNA. The process of removing the cross-linking is rough and possibly denatures the sample. There are also some that think the IP also pulls down targets naturally bound to ssDNA (but I would think in the minority). For all ChIP, the nanodrop was misleading for concentration. I now require dsDNA specific assays as initial QC/QA (picogreen, Qubit). If there is a question of the origin of the DNA on the second bioanalyzer traces, dilute some and PCR with the Illumina primers. You should only see an increase of the material with adapters.

01-11-2011, 08:15 AM	#6
obischof Member Location: france Join Date: Jan 2011 Posts: 11	We had no clusters that passed the internal QC of GA, though the first cycle was OK.