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Old 05-11-2016, 03:08 AM   #1
jpw39
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Default Unexpected peaks on Bioanalyzer trace, KAPA hyper prep for ChIP-seq library

Hi all,

Just wondering if anyone has observed high molecular weight peaks on their bioanlyzer traces for library preps - KAPA or otherwise. These were made from ChIP material with KAPA hyper prep kit, TruSeq adapters and KAPA's primers for amplification from P5/P7 sequences.

Thanks in advance...
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Old 05-11-2016, 05:00 AM   #2
nucacidhunter
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I wonder if you have any trace from input into library. Possible source of large fragments:
1- Suboptimal shearing of ChIP DNA
2- Anomaly in bioanalyser run due to library contaminants or run reagents

PCR would be less likely source of large fragments because polymerase will not be able to copy such large fragments
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Old 05-11-2016, 05:15 AM   #3
jpw39
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Thanks,

I suspect the input chromatin was not optimally sheared and I'm not surprised to see that the library material is quite a broad smear. However, the sharp peaks seen running larger than the 10kb marker are particularly strange to me, I can't think how the library prep would give rise to these.

A bioanalyzer anomaly seems more likely but I had 3 samples on the chip that had not been subjected to library prep which had no such peaks.
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Old 05-11-2016, 10:08 PM   #4
nucacidhunter
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If the library was run before clean up it will affect DNA migration speed on the Chip causing anomaly.

If you have left over from input material you can run them on the Chip and look for size distribution to see if larger fragments originate from input. The sharp peak could be unsheared chromatin mass.
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