Fragmentation of Long Reads Enables Reliable Mitogenome Assembly From Whole-Genome Amplification Data With Pervasive Palindromic Reads

Whole genome amplification (WGA), and in particular multiple displacement amplification (MDA), has become a key techniquefor genomic sequencing of microscopic organisms, yet it introduces artefacts such as palindromic (inverted chimeric) reads thatmay compromise downstream analyses. We assessed how pervasive palindromic reads generated by MDA impact the assemblyof tardigrade (Acutuncus giovanniniae and A. mecnuffi) mitogenomes sequenced with Oxford Nanopore technology. We showthat the MDA produces a high proportion of palindromic reads, often exceeding one-third of mitochondrial reads and frequentlyexhibiting complex multi-inversion structures. These artefacts severely impair long-read assembly, leading to low success ratesand inconsistent genome reconstruction. To solve this issue, a strategy based on in silico fragmentation of long reads into short,high-quality fragments, followed by short-read assembly, consistently produced complete and accurate circularised mitochondrialgenomes. Our results demonstrate that palindromic read formation can be, in some cases, a limitation of MDA coupled withlong-read sequencing, but this issue can be mitigated through read fragmentation. This approach provides a simple, robust andscalable solution for mitogenome assembly from data heavily affected by amplification artefacts, particularly in microscopic taxawhere whole genome amplification is often unavoidable.