Low concentration GA library

Melanie · 08-03-2010, 09:41 AM

Hi everyone,
I have just made my first library for sequencing. As i didn't have much material, after the initial gel purification I did 15 rounds of PCR, followed by a second gel purification as I had a lot of adapters coming through. My final library has the correct size distribution with no additional bands however the concentration is very low. The bioanalyser (high sensitivity DNA chip) results put it at 48.77pg/ul or 242.5pM but the q-PCR (KAPA sybr fast) results give me 140pg/ul or 710pM.

Is the difference that I see between the two quantitation methods due to the low amount of starting material?
Also should I amplify the library some more by doing another 3 rounds of PCR? Or is there enough there to send to the sequencers?

Any help would be greatly appreciated!

Thanks,

Melanie

GW_OK · 08-03-2010, 12:13 PM

I consider concentrations given by the bioanalyzer to be somewhat ballpark-ish but not worth much else. As long as you performed the qPCR correctly that is what I would believe. As far as dealing with low concentrations I have just recently been trying denaturation with 0.5N NaOH and then neutralizing with an equal volume of 0.5N HCl. After the neutralization you can dilute to whatever pM you want to load in 120ul of HT1. This seems to work pretty well for me anyway. I heard about it in a post somewhere on this forum, can't remember where.

NextGenSeq · 08-03-2010, 01:26 PM

You need ~10 picomoles to sequence per lane so it depends on what volume you have.

Melanie · 08-04-2010, 12:31 PM

Thanks!

I have talked with tech support at Illumina and here's what they said (in case anyone else has a similar problem or is just curious...)

"I would assume that the difference between the two quantitations is due to the low concentration as you suggest. I personally have more faith in the qPCR numbers assuming appropriate calibration has been done with that technique. In either case, the quantity of material sounds too low for moving into cluster generation which typically would require a 10 nM solution."

As I have only 30ul of sample, I'm going to repeat my protocol with more starting material to hopefully boost my end amount.

~Melanie

GW_OK · 08-04-2010, 02:34 PM

If you have 30ul at 710pM you could take 4ul, add 1ul 0.5N NaOH, wait 5 minutes, add 1ul 0.5N HCl, place on ice and then dilute 2ul of that in 118ul of HT1 for an 8pM ready-to-hyb aliquot.

I'm not saying you shouldn't start again I'm just pointing out that a low concentration library is not unload-able.

See also this thread

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08-03-2010, 09:41 AM	#1
Melanie Junior Member Location: New York, USA Join Date: May 2010 Posts: 6	Low concentration GA library Hi everyone, I have just made my first library for sequencing. As i didn't have much material, after the initial gel purification I did 15 rounds of PCR, followed by a second gel purification as I had a lot of adapters coming through. My final library has the correct size distribution with no additional bands however the concentration is very low. The bioanalyser (high sensitivity DNA chip) results put it at 48.77pg/ul or 242.5pM but the q-PCR (KAPA sybr fast) results give me 140pg/ul or 710pM. Is the difference that I see between the two quantitation methods due to the low amount of starting material? Also should I amplify the library some more by doing another 3 rounds of PCR? Or is there enough there to send to the sequencers? Any help would be greatly appreciated! Thanks, Melanie

08-03-2010, 12:13 PM	#2
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	I consider concentrations given by the bioanalyzer to be somewhat ballpark-ish but not worth much else. As long as you performed the qPCR correctly that is what I would believe. As far as dealing with low concentrations I have just recently been trying denaturation with 0.5N NaOH and then neutralizing with an equal volume of 0.5N HCl. After the neutralization you can dilute to whatever pM you want to load in 120ul of HT1. This seems to work pretty well for me anyway. I heard about it in a post somewhere on this forum, can't remember where.

08-03-2010, 01:26 PM	#3
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	You need ~10 picomoles to sequence per lane so it depends on what volume you have.

08-04-2010, 12:31 PM	#4
Melanie Junior Member Location: New York, USA Join Date: May 2010 Posts: 6	Thanks! I have talked with tech support at Illumina and here's what they said (in case anyone else has a similar problem or is just curious...) "I would assume that the difference between the two quantitations is due to the low concentration as you suggest. I personally have more faith in the qPCR numbers assuming appropriate calibration has been done with that technique. In either case, the quantity of material sounds too low for moving into cluster generation which typically would require a 10 nM solution." As I have only 30ul of sample, I'm going to repeat my protocol with more starting material to hopefully boost my end amount. ~Melanie

08-04-2010, 02:34 PM	#5
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	If you have 30ul at 710pM you could take 4ul, add 1ul 0.5N NaOH, wait 5 minutes, add 1ul 0.5N HCl, place on ice and then dilute 2ul of that in 118ul of HT1 for an 8pM ready-to-hyb aliquot. I'm not saying you shouldn't start again I'm just pointing out that a low concentration library is not unload-able. See also this thread