insert size question for sequencing

xp8 · 08-20-2011, 04:58 AM

-I use 75X2 sequencing to get a good coverage.
-Meanwhile, I want a good coverage of different size: inset size from 40bp to 300bp.

My question is:
For the 75X2 sequencing, Can I sequence 40bp insert (40 bp DNA fragment within 2 ends' adapter sequencing)?

(I assume that for >75bp insert, sequencing should be no problem, and
I do not care that for 75x2 sequencing of 40bp, the sequencing read may contain adapter-sequence).

Thanks
Victor

hbn · 08-25-2011, 07:16 AM

I think what is important here is the bridge amplification in the cluster formation.

I believe I heard/read somewhere that a total fragment length of 150 bp is the minimum for bridge formation (can anyone confirm this)? But with the Illumina adapters, I think your 40 bp insert will result in a total fragment size of about 150 bp (adapters are quite long), so I think that should not be a problem...

HESmith · 08-25-2011, 08:21 AM

Small inserts are not a problem, since adapter dimers are readily sequenced. I would recommend trimming the adapter sequences before starting any downstream analysis.

-Harold

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08-20-2011, 04:58 AM	#1
xp8 Junior Member Location: CT, USA Join Date: Jun 2011 Posts: 2	insert size question for sequencing -I use 75X2 sequencing to get a good coverage. -Meanwhile, I want a good coverage of different size: inset size from 40bp to 300bp. My question is: For the 75X2 sequencing, Can I sequence 40bp insert (40 bp DNA fragment within 2 ends' adapter sequencing)? (I assume that for >75bp insert, sequencing should be no problem, and I do not care that for 75x2 sequencing of 40bp, the sequencing read may contain adapter-sequence). Thanks Victor

08-25-2011, 07:16 AM	#2
hbn Member Location: Netherlands Join Date: Apr 2011 Posts: 16	I think what is important here is the bridge amplification in the cluster formation. I believe I heard/read somewhere that a total fragment length of 150 bp is the minimum for bridge formation (can anyone confirm this)? But with the Illumina adapters, I think your 40 bp insert will result in a total fragment size of about 150 bp (adapters are quite long), so I think that should not be a problem...

08-25-2011, 08:21 AM	#3
HESmith Senior Member Location: Bethesda MD Join Date: Oct 2009 Posts: 509	Small inserts are not a problem, since adapter dimers are readily sequenced. I would recommend trimming the adapter sequences before starting any downstream analysis. -Harold