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Hi everybody
I am using a GS Junior to sequence a rapid library of DNA fragments which start and end with an individual specific tag. Even though I measure the DNA concentration with Qubit and I carefully pool equimolar amounts of DNA from all individuals, I get large differences in number of reads per individual (up to 5fold). Also on the Bioanalyzer the DNA fragments look very similar in length distribution and concentration. I have performed two runs with the same four tags but different individuals and both times the tags GATATAGC and ATGTCGTA gave many reads and the tags TACAGATA and GAGCACTG gave 3-5 times less reads.
I found two papers in Plos One which describe variations in read numbers based on tag sequences but they contradict each other and the patterns they found do not match my findings: vanOrsouw et al., 2007 (says 5'CA tags are underrepresented, https://www.plosone.org/article/info...l.pone.0001172) and Binladen et al., 2007 (says 5'C tags are overrepresented, 5'T tags are underrepresented, https://www.plosone.org/article/info...l.pone.0000197).
Has anyone had similar experiences or does anyone have a possible explanation for this?
Thanks a lot in advance,
Joana
I am using a GS Junior to sequence a rapid library of DNA fragments which start and end with an individual specific tag. Even though I measure the DNA concentration with Qubit and I carefully pool equimolar amounts of DNA from all individuals, I get large differences in number of reads per individual (up to 5fold). Also on the Bioanalyzer the DNA fragments look very similar in length distribution and concentration. I have performed two runs with the same four tags but different individuals and both times the tags GATATAGC and ATGTCGTA gave many reads and the tags TACAGATA and GAGCACTG gave 3-5 times less reads.
I found two papers in Plos One which describe variations in read numbers based on tag sequences but they contradict each other and the patterns they found do not match my findings: vanOrsouw et al., 2007 (says 5'CA tags are underrepresented, https://www.plosone.org/article/info...l.pone.0001172) and Binladen et al., 2007 (says 5'C tags are overrepresented, 5'T tags are underrepresented, https://www.plosone.org/article/info...l.pone.0000197).
Has anyone had similar experiences or does anyone have a possible explanation for this?
Thanks a lot in advance,
Joana