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Old 02-02-2015, 07:23 AM   #1
cknox
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Location: Madison, WI

Join Date: Jul 2012
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Default NxSeq Long Mate Pair Technology for up to 20 kb mate pair libraries

Hello SEQanswers users,
Lucigen would like to announce the availability of our new NxSeq Long Mate Pair library kits and services. The product enables you to create the longest and most efficient mate pair libraries for your genome to date. With a bead-based size selection method, you can obtain tight distribution of a user selected insert size from 2 to 8 kb, and with a newly released gel-based size selection protocol, you can consistently obtain mate pair libraries with up to 20 kb insert sizes.

Bioinformatically, long span mate pair libraries enable you to accomplish the same type of assembly results that you would with a Pac Bio long read without the need to access a new instrument, as our technology runs on existing Illumina machines.

You can find out more information about the product here: http://lucigen.com/NxSeq-Long-Mate-Pair-Library-Kit/

In addition, you can find supporting poster data here: http://lucigen.com/docs/posters/NxSe...-Mate-Pair.pdf

Construction of these libraries are also available as a service through Lucigen. For more information about our genome closure services, click here: http://lucigen.com/next-gen-sequenci...-services.html

Please do not hesitate to contact Lucigen with any questions you may have.
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Old 07-15-2015, 09:58 AM   #2
jpmirandam
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Location: Santiago, Chile

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Dear cknox,

I bought mate pair kits, both for illumina and ion torrent in order to prepare 8 kb insert libraries.

None of the protocols has worked for us and we lost more than 98% of the DNA before adaptor ligation and purification using AMPureXP (Our AMPureXP work correctly for other protocols).

After size selection, almost 100% of remaining DNA is recovered, but not enough to continue with coupler ligation (lower than 150 ng).

I wonder if other researchers have reported these problems and if you have suggestions to go ahead with the protocol.

Best regards,

JP.
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Old 08-12-2015, 02:05 PM   #3
Lucigen_NGS_Service
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Hello Dr. Miranda and thank you for your inquiry. In our follow-up with you, you noted the primary DNA loss occurred after the adaptor ligation (instead of before as stated in your initial post), which is atypical for users of either the long mate pair or ion torrent kits. While some loss during the cleanup of the adaptor ligation is normal and expected, yours is the first report of losses to that high an extent. We identified that it may of help to check your shearing on g-TUBES (Covaris) prior to ligating adaptors, as we have noted that these devices perform as expected provided you normalize masses to those in the manufacturer’s table, but the mass you chose of 11.5 mcg is between the 8 and 15 mcg settings. In cases that fall outside, it is best to test the settings used to see if all liquid passes through the device, and once done, what the sheared DNA looks like on a gel. We have encountered higher DNA loss in the adaptor ligation cleanup when DNA shearing was incomplete due to sample volume not completely passing through the device. We welcome any follow-up discussion or questions in either this forum or we can always be reached at [email protected].
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