Best Way to make Mate-Pair library?

[Manna]

What do people think is the best way to make a mate-pair library? I need to do some with 3 and/or 6 kb inserts and probably at least one with a larger 10kb insert. Are there any consistent problems with the Nextera kit? How do the data you get from the Nextera kit compare with other kits? What's the word on the street?

Thanks!
m

[ychang]

we have succeed make 4-7kb mate-pair lib with Nextera kit, and will try 10kb this week.

[aammar]

Hi ychang,

I just started using the Nextera MP kit. How did you manage that short of kb insert? I get them on the larger side. I ideally want 3-4 kb insert size. Suggestions? Thank you in advance!

[jjcooper]

Hi ychang,
Did you have any luck with the 10 kb prep?
Hi aammar,
What size do your insert sizes usually end up? are you using the gel-plus protocol?
Thanks

[MLog]

Hi ychang (and everyone who has experience with Nextera MP, too),
did you already sequenced and mapped these libraries? I wonder how many reads mapped with expected insert size?
I tried Nextera MP for the first time, but the results are a bit disappointing - only ~ 40% of reads map in correct orientation and with expected insert size. The rest 60% seem to be short fragments that yield standard paired-end reads.

[Ama]

Hi

@MLog: I assume you removed the junction adapters of the nextera data before you did the mapping, can you please let me know what tools/approach you used for that? I been following the technical notes to trim the adapters off and to identify the reads orientation RF/FR, but the results I am seeing are very disappointing. So any help would be much appreciated.
Thanks

[jjcooper]

@MLog,
Did you do the gel-free or gel-plus protocol? How did your QCs with the Bioanalyzer look?

[syfo]

You might also need to reverse complement the reads before mapping them because some aligners expect the mates to be in a convergent orientation (head-to-head).

[sara_ahmed]

What is the circularization efficiency using the Nextera MP protocol? Is there a way to assess that in the protocol?

[hfaoro]

Hi,
About nextera MP protocol, does anyone knows if is it possible to use covaris microTUBE with fiber, instead of T6 tube, to shear the DNA after circularization step?

[BobS]

hfaoro , we used to use microTubes also.
Covaris Fragmentation for about 400bp fragments: For each 300ul + 9ul + 12ul reaction add 80 ul TE to bring volume up to near 400 ul microTube. Load 3 , 6 x 16 mm MicroTubes per sample 3 x 130ul = 390ul

Covaris E210 settings for ~400 bp fragments
5% Duty Cycle, 3 Intensity, 200 Cycles per burst, 110 sec. duration water cooled at 6 - 8 deg C. You probably will have to tweak your settings.