Carvalho, A. B.; Kim, B. Y.; Uno, F.

We recently showed that Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) have very strong sequencing bias against simple satellites, probably caused by single-stranded DNA folding into non-canonical (non-B) structures during sequencing. Here we extend these observations by computational and experimental approaches in the Drosophila and human genomes. We found that (i) only a small subset of simple satellites cause sequencing bias; many satellites (e.g., (ACTGGG)n ) are benign and easily sequenced. (ii) The biases most likely are caused by two distinct non-B DNA structures: triplex DNA formed by some, but not all, AG-rich satellites (only those predicted to form strong mirror repeats), and hairpins formed by some, but not all, AT-rich satellites (only those predicted to form fairly strong inverted repeats). (iii) The correlation between the predicted stability of these non-B structures, and the strength of sequencing bias indicates that non-B DNA is indeed the culprit. (iv) The likely source of these non-B structures is single-stranded DNA formed during ONT and PacBio sequencing, and hence its removal might solve the bias. We tested this by adding single-strand binding protein to ONT sequencing, and found that it irreversibly kills the flow cells. (v) A recent sequencing effort in Drosophila melanogaster using very high depth Ultra-Long ONT sequencing (967x) still failed to assemble many genes located near satellite blocks. Brute-force will not solve the problem; instead, further investment is needed by the sequencing companies to achieve truly unbiased sequencing.