Adapter Dimer Contamination Irregularities Between Multiplexed Samples [Chip-Seq/Rna-Seq]

Question

[Sukhi Singh]

Hi guys,

Whenever I start working with new samples (multiplexed on HiSeq-2000), some samples house adapter-dimer contaminations and others dont. Samples are prepared by same person using same protocol, sequenced at same time in the same machine in same/different lanes, so why this difference. To my understanding the 45° Y shaped adapters are ligated to the DNA fragment, gel purified and then index + multiplex adapters are ligated which then are PCR amplified and after that ligated on to the cluster bed for bridge amplification. So, the adapters are also amplified and should present in all of them or may be I am missing something.

Thanks

Source : http://genome.med.harvard.edu/

Answer 1

[Sean Davis]

Excessive adapter contamination can be associated with limited DNA input quantities. You may want to look at the amount of input DNA that was used to see if there is a correlation. That said, adapter contamination may or may not constitute a real problem with the data; in other words, the data may still be useable after cleaning up the contamination (often alignment will do the trick).

[Sukhi Singh]

I got your point, the contamination is inversely proportional the starting material, but still all of them have adapters ligated, still some of them show it and some don't. If we have an ideal starting material, still we amplify the DNA and should observe the cotamination though less!! What do you think.

Cheers

[Sean Davis]

The adapters are on every sequence, but they should not be sequenced unless the insert size is small. In that case, the ends of reads will contain adapters. For that reason, there are many softwares for removing adapters from the ends of reads.

[Sukhi Singh]

Thanks Sean, clears things. Also, this post was helpful as well about insert size and adapters.

Answer 2

[qliu]

Have you considered clean-up or size selection before PCR? Even though the software tools can help remove the contamination data, A proper DNA sizing or cleanup can help improve the sequencing efficiency and the data quality. Check https://www.nvigen.com/dna-size-selection-tutorial/.