Comment on "DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification"

Abstract

Chen et al (Reports, 17 February 2017, p. 752) highlight an important problem of sequencing artifacts caused by DNA damage at the time of sample processing. However, their manuscript contains several errors that led the authors to incorrect conclusions. Moreover, the same sequencing artifacts were previously described and mitigated in The Cancer Genome Atlas and other published sequencing projects.

Figure 1.

(A) Picard oxoQ score vs. GIV_{G_T} for &1,900 TCGA tumor exomes. Lack of agreement between oxoQ and GIV_{G_T} scores is apparent in samples with high DNA damage (oxoQ < 30) highlighted in the gray box. GIV_{G_T} is calculated with Chen et al.’s code (estimate_damage.pl) with default parameters; oxoQ was calculated with Picard CollectSequencingArtifactMetrics (11) output. (B) OxoQ vs. GIV_{G_T} corrected (using base quality Q > 20 and coverage depth ≥ 20, comparable to Picard defaults) showing excellent agreement. (C) Histogram of individual base qualities in a typical TCGA 8-oxoG damaged sample (oxoQ = 24.2) compared to a low-damage sample (oxoQ=48.9). Most of the bases in the damaged sample have Q < 30 and hence are ignored by default GIV_{G_T}. This explains both the lack of agreement between the scores seen in (A) and authors missing the sequence context associated with 8-oxoG damage (D vs E) –– the Q > 30 filtering removed bases with the type of damage that is being quantified. “Lego” plot showing the distribution of errors in different 5′ and 3′ sequence contexts (plotted in reverse complement by convention), with Chen et al.’s (D) and our corrected (E) filtering criteria, showing distortion from the characteristic 8-oxoG context pattern arising from data selection criteria. (F) The fraction of bases that remain after applying Chen et al.’s filters show that most bases are removed in highly damaged samples.

Figure 2.

Comparison of 8-oxoG related error rates and other error modes. Samples (A-C) with corrected GIV_{G_T} damage levels below 5 (equivalent to oxoQ & 35) have fewer 8-oxoG–related base errors (red) compared to other sequencing error modes (blue). (A) Count of tumors with a specific corrected GIV_{G_T} score (majority of TCGA samples have very low damage levels). (B) Percentage of additional base mismatches caused by 8-oxoG in relation to GIV_{G_T} score. (C) 8-oxoG–related error rates (red) and other error modes (blue). Other modes dominate until corrected GIV_{G_T} & 5. (D) Copy of Fig 4E from Chen et al. (1). (E) Our estimated FDR vs. corrected GIV_{G_T} for &1,900 TCGA tumors from MuTect (14) before 8-oxoG filtering (GIV_{G_T} < 7.5). (F, G) Estimated FDR vs. corrected GIV_{G_T} for &1,900 TCGA tumors from MuTect before (F) and after (G) 8-oxoG filtering (as described in (2)). Results demonstrate no inflation in number of mutation calls after applying the 8-oxoG software filter.