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I'm currently working with DNA samples originating from museum specimens, this means they have been stored in formaldehyde for the last 50-100 years.

The DNA I'm analysing has been sequenced by Illumina NovaSeq 6000 with paired-end sequencing.

As far as I'm aware NovaSeq uses two-colour chemistry, where Green is T, red is C, yellow (green + red) is A, and no green, no red is G. Over time and increase in sequencing cycles the green and red signal will become weaker, resulting in mistaken categorization of the nucleotides as G in the tails of reads.

This poly-G pattern is however only observed in the 3' end of the 2nd read in the pairs, which I find a bit weird.

Is this an expected pattern (only in the 2nd read in a pair) from NovaSeq or could this possibly also have something to do with my samples being stored in formaldehyde for the last 100 years, thus degrading or affecting the DNA?

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  • $\begingroup$ Have you tried sequencing some fresh sample to see if the same pattern appears? $\endgroup$ – MattDMo Dec 29 '20 at 17:40
  • $\begingroup$ unfortunately im not the one performing the sequencing, im doing the bioinformatic analysis. But yes some newer samples has been sequenced, however this has been done with Illumina Hiseq4000 and these samples does not show the same pattern with Poly-G. But the Hiseq uses a four colour scheme. $\endgroup$ – RAHenriksen Dec 30 '20 at 9:24
  • $\begingroup$ of course ideally the newer samples would also have been done with NovaSeq, to determine if it is the sequencing or the sample ages. $\endgroup$ – RAHenriksen Dec 30 '20 at 9:26
  • $\begingroup$ Is this from a single sequencing run or multiple sequencing runs? I would suspect that something got jacked on the sequencer if I saw that in my data. Did you monitor the sequencing run? What do the quality scores look like? Have you run FASTQC or FASTP? I would recommend investigating all of those things. $\endgroup$ – Maximilian Press Dec 30 '20 at 18:28

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