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Hello everyone,
I've got two questions concerning the detection of structural variations:
1.) We sequenced the same cell line twice with the Illumina GA II (paired-end, 100bp read length), but with different insert sizes; ca. 400bp in run1 and 200bp in run2 (ends may overlap here). Kinda strange thing happend here when we detected deletions: In run1 we detected deletions (ca. 1.500) of various sizes (ca. 150bp - 200.000bp), quite a lot of deletions of a few thousand basepairs. In run2 almost 100% of the deletions ranged between only 100bp and 1.000bp. We saw this using Breakdancer and GASV using the same parameters and filtering criteria in both runs. The sequence and fragment coverage is also comparable in both runs. I don't quite understand why there are such big differences and some kind of upper limit in run2. Maybe some of you have experienced something similar.
2.) Does anyone know an effect during the paired-end sequencing process (Illumina GA II in my case) that can cause artificial inversions? We noticed quite strong deviations in the number of detected inversions across different experiments or samples (we used GASV for variant detection); it ranged from 10 to 20.000 inversions using the same programs and parameters during the analysis. I'd never expect that much inversions and such discrepancies even across different samples. Does someone know if that might be a technical problem in the lab or in the sequencing machine?
I'd be very glad if someone had some ideas, that help me understand these effects. Thanks in advance.
Cheers,
Christoph
I've got two questions concerning the detection of structural variations:
1.) We sequenced the same cell line twice with the Illumina GA II (paired-end, 100bp read length), but with different insert sizes; ca. 400bp in run1 and 200bp in run2 (ends may overlap here). Kinda strange thing happend here when we detected deletions: In run1 we detected deletions (ca. 1.500) of various sizes (ca. 150bp - 200.000bp), quite a lot of deletions of a few thousand basepairs. In run2 almost 100% of the deletions ranged between only 100bp and 1.000bp. We saw this using Breakdancer and GASV using the same parameters and filtering criteria in both runs. The sequence and fragment coverage is also comparable in both runs. I don't quite understand why there are such big differences and some kind of upper limit in run2. Maybe some of you have experienced something similar.
2.) Does anyone know an effect during the paired-end sequencing process (Illumina GA II in my case) that can cause artificial inversions? We noticed quite strong deviations in the number of detected inversions across different experiments or samples (we used GASV for variant detection); it ranged from 10 to 20.000 inversions using the same programs and parameters during the analysis. I'd never expect that much inversions and such discrepancies even across different samples. Does someone know if that might be a technical problem in the lab or in the sequencing machine?
I'd be very glad if someone had some ideas, that help me understand these effects. Thanks in advance.
Cheers,
Christoph