An improved protocol for sequencing of repetitive genomic regions and structural variations using mutagenesis and next generation sequencing.
The rise of Next Generation Sequencing (NGS) technologies has transformed de novo genome sequencing into an accessible research tool, but obtaining high quality eukaryotic genome assemblies remains a challenge, mostly due to the abundance of repetitive elements. These also make it difficult to study nucleotide polymorphism in repetitive regions, including certain types of structural variations. One solution proposed for resolving such regions is Sequence Assembly aided by Mutagenesis (SAM), which relies on the fact that introducing enough random mutations breaks the repetitive structure, making assembly possible. Sequencing many different mutated copies permits the sequence of the repetitive region to be inferred by consensus methods. However, this approach relies on molecular cloning in order to isolate and amplify individual mutant copies, making it hard to scale-up the approach for use in conjunction with high-throughput sequencing technologies. To address this problem, we propose NG-SAM, a modified version of the SAM protocol that relies on PCR and dilution steps only, coupled to a NGS workflow. NG-SAM therefore has the potential to be scaled-up, e.g. using emerging microfluidics technologies. We built a realistic simulation pipeline to study the feasibility of NG-SAM, and our results suggest that under appropriate experimental conditions the approach might be successfully put into practice. Moreover, our simulations suggest that NG-SAM is capable of reconstructing robustly a wide range of potential target sequences of varying lengths and repetitive structures.
European Bioinformatics Institute, EMBL-EBI, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom. sbotond@ebi.ac.uk
Botond
B
Sipos
Tim
T
Massingham
Adrian M
AM
Stütz
Nick
N
Goldman
D000818:Animals; D001483:Base Sequence; D003198:Computer Simulation; D005240:Feasibility Studies; D016678:Genome / Q000235:genetics*; D023281:Genomics / Q000379:methods; D006801:Humans; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*