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Cited by 16

The DOI matches its Crossref record; 34 of the 34 references it cites were found in a registry or an index.

Microsatellite Length Scoring by Single Molecule Real Time Sequencing – Effects of Sequence Structure and PCR Regime

Liljegren, Mikkel Meyn, de Muinck, Eric Jacques, Trosvik, Pål·2016·PLOS ONE
DOI 10.1371/journal.pone.0159232PMC PMC4945053
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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.

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Crossref · 10.1371/journal.pone.0159232

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Figures

Fig 1 Mean distribution of enzyme slippage errors. Each dot in the graphs is a proportion of reads (y-axes) corresponding to the microsatellite lengths (bp) indicated on the x-axes. The dots are color coded according to the number of PCR cycles indicated above. A. Mean of two replicates of 30xCA repeat sequence. B. Mean of two replicates of 20xATCT repeat sequence. C. Mean of four replicates of 10xATCT repeat sequence. Singleton reads and reads with lengths not corresponding to multiples of the repeat units are not shown.
Fig 1 Mean distribution of enzyme slippage errors. Each dot in the graphs is a proportion of reads (y-axes) corresponding to the microsatellite lengths (bp) indicated on the x-axes. The dots are color coded according to the number of PCR cycles indicated above. A. Mean of two replicates of 30xCA repeat sequence. B. Mean of two replicates of 20xATCT repeat sequence. C. Mean of four replicates of 10xATCT repeat sequence. Singleton reads and reads with lengths not corresponding to multiples of the repeat units are not shown.CC BY · PubMed Central
Fig 2 Microsatellite length distributions for no-PCR controls. A. 30xCA. B. 20xATCT. C. 10xATCT (mean of scores from constructs 1 and 2). The y-axes indicate the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axes. Singleton read lengths have been omitted from the plots for enhanced visualization (see S4, S9, S14 and S19 Figs for full details).
Fig 2 Microsatellite length distributions for no-PCR controls. A. 30xCA. B. 20xATCT. C. 10xATCT (mean of scores from constructs 1 and 2). The y-axes indicate the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axes. Singleton read lengths have been omitted from the plots for enhanced visualization (see S4, S9, S14 and S19 Figs for full details).CC BY · PubMed Central
Fig 3 Mean microsatellite length distributions for PCR amplified fragments. A, D, G and J: 30xCA. B, E, H and K: 20xATCT. C, F, I and L: 10xATCT. A-C: 10 PCR cycles. D-F: 20 PCR cycles. G-I: 30 PCR cycles. J-L: 40 PCR cycles. All panels show mean results from two replicate PCR reactions. The y-axes indicate the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axes. In each panel the target sequence length can be seen to dominate. Singleton read lengths have been omitted from the plots for enhanced visualization (see S5–S8, S10–S13, S15–S18 and S20–S23 Figs for full details).
Fig 3 Mean microsatellite length distributions for PCR amplified fragments. A, D, G and J: 30xCA. B, E, H and K: 20xATCT. C, F, I and L: 10xATCT. A-C: 10 PCR cycles. D-F: 20 PCR cycles. G-I: 30 PCR cycles. J-L: 40 PCR cycles. All panels show mean results from two replicate PCR reactions. The y-axes indicate the number of SMRT sequence reads corresponding to the sequence length (bp) indicated on the x-axes. In each panel the target sequence length can be seen to dominate. Singleton read lengths have been omitted from the plots for enhanced visualization (see S5–S8, S10–S13, S15–S18 and S20–S23 Figs for full details).CC BY · PubMed Central
Fig 4 Microsatellite length scoring accuracy. A. Percentage of reads matching the expected sequence length. B. Shannon entropy of measured microsatellite lengths in each experiment. The plotted values are based on both stepwise and non-stepwise errors.
Fig 4 Microsatellite length scoring accuracy. A. Percentage of reads matching the expected sequence length. B. Shannon entropy of measured microsatellite lengths in each experiment. The plotted values are based on both stepwise and non-stepwise errors.CC BY · PubMed Central
Fig 5 Directionality of indel mutation events. Percentages of non-target read lengths are on the y-axes, while the microsatellite structures and directionality of mutation events (I = insertion; D = deletion) are indicated on the x-axes. The boxes represent the inter-quartile range with the thick lines representing the medians. The whiskers are 1.5 times the inter-quartile range and outliers are plotted as individual dots. A. Stepwise mutations by integer numbers of repeat units. B. Non-stepwise mutations. C. Indel mutations by one or more bp in linker sequences.
Fig 5 Directionality of indel mutation events. Percentages of non-target read lengths are on the y-axes, while the microsatellite structures and directionality of mutation events (I = insertion; D = deletion) are indicated on the x-axes. The boxes represent the inter-quartile range with the thick lines representing the medians. The whiskers are 1.5 times the inter-quartile range and outliers are plotted as individual dots. A. Stepwise mutations by integer numbers of repeat units. B. Non-stepwise mutations. C. Indel mutations by one or more bp in linker sequences.CC BY · PubMed Central

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Metadata

Authors
Liljegren, M. M., de Muinck, E. J., & Trosvik, P.
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2016-07-14
Volume
11
Issue
7
Pages
e0159232
ISSN
1932-6203
DOI
10.1371/journal.pone.0159232
PMC
PMC4945053
Cited by
16
References
34
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
Norges Forskningsråd (214285/F20)

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