This is my first time in using the TruSeq library Nano LT preparation kit, and also I was using ezRAD method, digesting the DNA samples using Ecor I and then followed the protocol in the TruSeq Nano Kit. However, the final library fragments are much larger, 600-1100 bp, than expected 500-600 bp. Any suggestions on how to improve the method? Anyone have any experiences in getting larger fragment sizes when using the new TruSeq Nano kit? Thank you so much.
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Last edited by Dongmei_MPI; 10-02-2016, 01:57 AM. -
We are using the nano kit but with a sonication step. Our fragments size are also in your range (600 to 1000bp). But it's not an issue for us as we are doing 2*250nt runs.
Did you try the 350bp insert size option? Maybe you will end up with more suitable sizes. -
enzymatic fragmentation is controlled by the ratio of DNA to enzyme. bigger frags than you want mean too much DNA/not enough enzymeMicrobial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.Comment
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550 bp insert option
To huguesparri: I selected the 550 bp insert option. there are two options, one is for 250 and 550 bp insert options. I am planning to run 2x 300 bp PE sequencing. To me, if you do 2x250 nt run, will your insert of 600-1000 bp too larger?Comment
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If you are more interested in the smaller size range of your library, keep in mind that the smaller fragments will cluster more efficiently than the larger fragments anyway, so you'll see more sequences in the ~600bp range than the ~1100bp range.Comment
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