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Old 08-16-2016, 03:47 PM   #1
meriem
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Question library preparation and removing adapters

hello,
i'm new user of illumina miseq technology and i'm finding some difficulties understanding some basics.
if i select in the machine that i want to get fragments of 250 bp why do i still get fragments from 35 bp to 250 bp in my output? ( i don't know how the fragmentation step is done, if the transposon generate only the fragments of 250 bP or different sizes)
and does the adapters used are automaticaly removed from the fragments in the output files?

could someone help me to understand
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Old 08-16-2016, 07:44 PM   #2
idedios
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So a few things:

You get fragments ranging from 0 to 250bp because those fragments are present in the final library. If you run a bioanalyzer trace you should see all the fragments with some tailing off at 1kb. Generally this is seen in all sequencing-by-synthesis assays where random shearing is used and AmpureXP beads are used to remove most fragments <100bp.

By the way what kind of assay are you running?
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Old 08-17-2016, 05:34 AM   #3
meriem
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thank you for you reply, i'm doing microbial DNA sequencing using nextera XT kit, but i'm still confused when we say 250 bp is that with adapters or only the fragments of the sample, and how can i know if i still have the adapters in my sequences?
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Old 08-17-2016, 02:55 PM   #4
nucacidhunter
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Quote:
Originally Posted by meriem View Post
thank you for you reply, i'm doing microbial DNA sequencing using nextera XT kit, but i'm still confused when we say 250 bp is that with adapters or only the fragments of the sample, and how can i know if i still have the adapters in my sequences?
Machine will do number of sequencing cycles set on the run regardless of fragment size. Sequencer lacks any mechanism for estimation of fragment length. If you are running 2x250, only library inserts shorter than 250 bp will have adapter sequences in their 3' end.

You can find reads with adapters by searching for Nextera adapter sequences.
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