Abstract
Microsatellites are DNA sequences consisting of repeated, short (1–6 bp) sequence motifs that are highly mutable by enzymatic slippage during replication. Due to their high intrinsic variability, microsatellites have important applications in population genetics, forensics, genome mapping, as well as cancer diagnostics and prognosis. The current analytical standard for microsatellites is based on length scoring by high precision electrophoresis, but due to increasing efficiency next-generation sequencing techniques may provide a viable alternative. Here, we evaluated single molecule real time (SMRT) sequencing, implemented in the PacBio series of sequencing apparatuses, as a means of microsatellite length scoring. To this end we carried out multiplexed SMRT sequencing of plasmid-carried artificial microsatellites of varying structure under different pre-sequencing PCR regimes. For each repeat structure, reads corresponding to the target length dominated. We found that pre-sequencing amplification had large effects on scoring accuracy and error distribution relative to controls, but that the effects of the number of amplification cycles were generally weak. In line with expectations enzymatic slippage decreased proportionally with microsatellite repeat unit length and increased with repetition number. Finally, we determined directional mutation trends, showing that PCR and SMRT sequencing introduced consistent but opposing error patterns in contraction and expansion of the microsatellites on the repeat motif and single nucleotide level.
Highlights
Microsatellite DNA, known as short tandem repeats (STR) or simple sequence repeats, are tracts of mono- to hexanucleotide repeats of variable length that are widely distributed in the genomes of animals, plants and microbes [1]
DNA polymerase slippage was more pronounced in the dinucleotide repeat sequence than the tetranucleotide, and more mutations were observed in the longer tetranuclotide sequence than the shorter
The mutation rates we observed, with the highest rates occurring in the dinucleotide repeat sequence, and higher rates in the longer tetranuclotide sequence than the shorter, are in line with previous studies of variability of naturally occurring microsatellite alleles in genomes [27,28,29]
Summary
Microsatellite DNA, known as short tandem repeats (STR) or simple sequence repeats, are tracts of mono- to hexanucleotide repeats of variable length that are widely distributed in the genomes of animals, plants and microbes [1]. Microsatellite evolution is commonly described by a stepwise mutation model [3], in which transient dissociation of two replicating DNA strands leads to looping by one or more repeat units [4]. The looping of one strand, with subsequent misaligned re-association, leads to enzymatic slippage resulting in microsatellite expansion if looping occurs on the nascent strand and contraction if it occurs on the template. Many details concerning the microsatellite mutation process are still unclear, there is experimental evidence that DNA polymerase activity is the main mechanism [5,6]
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