I just received sequencing results of small RNA fraction (21-25nt) sequenced with HISeq and got puzzling results. Many of the sequences map to known small RNAs, but the mapping is on both strands, whereas the library was prepared as strand-specific.
Summary of library preparation:
(1) Small RNA fraction was isolated
(2) ligation to 3' adapter
(3) ligation to 5' adapter
(4) Reverse-transcription with specific primer
(5) all other steps, finally sequencing of 40nt.
Due to the specific ligation of the 5' adapter to the 5' end of the RNA I can't see how the library has lost its strand specificity completely (about 1:1 ratio).
Has anyone had a similar experience with such lack of "strandiness" with strand-specific library?
Summary of library preparation:
(1) Small RNA fraction was isolated
(2) ligation to 3' adapter
(3) ligation to 5' adapter
(4) Reverse-transcription with specific primer
(5) all other steps, finally sequencing of 40nt.
Due to the specific ligation of the 5' adapter to the 5' end of the RNA I can't see how the library has lost its strand specificity completely (about 1:1 ratio).
Has anyone had a similar experience with such lack of "strandiness" with strand-specific library?
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