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Preferential amplification of repetitive DNA during whole genome sequencing library creation from historic samples

Star, Bastiaan, Hansen, Marianne HS, Skage, Morten, Bradbury, Ian R., Godiksen, Jane A., Kjesbu, Olav S., Jentoft, Sissel·2016·STAR: Science & Technology of Archaeological Research
DOI 10.1080/20548923.2016.1160594
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Image 1
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Image 2
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Figure 1 Dinucleotide repeat content in vertebrate assemblies and Atlantic cod sequencing data. (a) The amount of AC and AG dinucleotide repeats was obtained using Tandem Repeat Finder (TRF, version 407b) in 65 vertebrate genome assemblies (Ensembl, release 76), including salmon and rainbow trout. Estimates were divided by total assembly size, excluding undetermined bases. Assemblies with more extreme repeat content are indicated (see Supplementary Table 2 for a detailed species list). (b) Dinucleotide repeat estimates in Atlantic cod sequencing data from contemporary (red), historic Norwegian (grey) and historic Canadian (black) samples. Each dot represents a single library, generated from a different individual. The repeat content in the Atlantic cod assembly (red, star) is indicated for comparison. The average fragment length (bp) is shown for the historic Norwegian and Canadian samples. Notice the different scales on the x- and y- axes for each panel.
Figure 1 Dinucleotide repeat content in vertebrate assemblies and Atlantic cod sequencing data. (a) The amount of AC and AG dinucleotide repeats was obtained using Tandem Repeat Finder (TRF, version 407b) in 65 vertebrate genome assemblies (Ensembl, release 76), including salmon and rainbow trout. Estimates were divided by total assembly size, excluding undetermined bases. Assemblies with more extreme repeat content are indicated (see Supplementary Table 2 for a detailed species list). (b) Dinucleotide repeat estimates in Atlantic cod sequencing data from contemporary (red), historic Norwegian (grey) and historic Canadian (black) samples. Each dot represents a single library, generated from a different individual. The repeat content in the Atlantic cod assembly (red, star) is indicated for comparison. The average fragment length (bp) is shown for the historic Norwegian and Canadian samples. Notice the different scales on the x- and y- axes for each panel.CC-BY · From the PDF
Figure 2 Effect of ampli fi cation conditions on repeat proliferation and endogenous DNA content. The proportion of AC (yellow ) , AG (blue) dinucleotide repeats was obtained using TRF. Reads aligning with a MapQ value of 25 or higher toward the Atlantic cod assembly are considered endogenous (black). A total of six ligated extracts from Canadian specimens were ampli fi ed either 12 or 18 PCR cycles, yielding 12 separate sequencing libraries (see text for details). Lines connect libraries that were created using the same extract. Notice the different scale on the y-axes for each panel. For libraries ampli fi ed using 18 cycles, an overestimation of fragment length and a multimodal distribution of BioAnalyzer plots indicated the presence of heteroduplexes, which is suggestive of reaching the plateau phase of PCR during library ampli fi cation.
Figure 2 Effect of ampli fi cation conditions on repeat proliferation and endogenous DNA content. The proportion of AC (yellow ) , AG (blue) dinucleotide repeats was obtained using TRF. Reads aligning with a MapQ value of 25 or higher toward the Atlantic cod assembly are considered endogenous (black). A total of six ligated extracts from Canadian specimens were ampli fi ed either 12 or 18 PCR cycles, yielding 12 separate sequencing libraries (see text for details). Lines connect libraries that were created using the same extract. Notice the different scale on the y-axes for each panel. For libraries ampli fi ed using 18 cycles, an overestimation of fragment length and a multimodal distribution of BioAnalyzer plots indicated the presence of heteroduplexes, which is suggestive of reaching the plateau phase of PCR during library ampli fi cation.CC-BY · From the PDF
Image 5
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Image 6
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Figure 4 Dinucleotide repeat (AC and AG) bias in contemporary human sequencing data. (a) AC and AG repeat content after ampli fi cation with ten different polymerase-buffer systems compared to the content of the original, unampli fi ed library (dashed red line). The number on the x-axis shows the number of PCR cycles. We only used sequencing data with a minimum of 100,000 reads per combination of polymerase type and cycle number. (b) A higher resolution of the AC and AG content of a sequencing library ampli fi ed with Herculase II Fusion (grey) and Phusion HF (purple) compared to the original, unampli fi ed library (dashed red line). PCR reactions were performed at 2-cycle intervals from 20 to 40 cycles. The data in panel (a) and (b) were previously analyzed for GC and length bias in Dabney and Meyer (2012). Notice the different scale on the y-axes for each panel.
Figure 4 Dinucleotide repeat (AC and AG) bias in contemporary human sequencing data. (a) AC and AG repeat content after ampli fi cation with ten different polymerase-buffer systems compared to the content of the original, unampli fi ed library (dashed red line). The number on the x-axis shows the number of PCR cycles. We only used sequencing data with a minimum of 100,000 reads per combination of polymerase type and cycle number. (b) A higher resolution of the AC and AG content of a sequencing library ampli fi ed with Herculase II Fusion (grey) and Phusion HF (purple) compared to the original, unampli fi ed library (dashed red line). PCR reactions were performed at 2-cycle intervals from 20 to 40 cycles. The data in panel (a) and (b) were previously analyzed for GC and length bias in Dabney and Meyer (2012). Notice the different scale on the y-axes for each panel.CC-BY · From the PDF
Figure 5 Hypothetical mechanism of self-priming of dinucleotide repeats during the ampli fi cation of whole genome sequencing libraries. (a) Single stranded repetitive DNA (red) anneals simultaneously with the library speci fi c ampli fi cation primer (grey). Elongation from the primer is blocked by repetitive DNA, which is downstream annealed, leading to the formation of two fragments. (b) Annealing and elongation from ampli fi cation primer or repetitive DNA generates a population of repeatassociated primers with speci fi c af fi nity for repetitive DNA. (c) Annealing and hybridization of complementary repetitive regions generates fragments with P5 and P7 primer pairs suitable for sequencing. Alternatively, repeat-associated primers with af fi nity for repetitive regions speci fi cally amplify those regions. The size of primer and repeat regions is not shown to scale.
Figure 5 Hypothetical mechanism of self-priming of dinucleotide repeats during the ampli fi cation of whole genome sequencing libraries. (a) Single stranded repetitive DNA (red) anneals simultaneously with the library speci fi c ampli fi cation primer (grey). Elongation from the primer is blocked by repetitive DNA, which is downstream annealed, leading to the formation of two fragments. (b) Annealing and elongation from ampli fi cation primer or repetitive DNA generates a population of repeatassociated primers with speci fi c af fi nity for repetitive DNA. (c) Annealing and hybridization of complementary repetitive regions generates fragments with P5 and P7 primer pairs suitable for sequencing. Alternatively, repeat-associated primers with af fi nity for repetitive regions speci fi cally amplify those regions. The size of primer and repeat regions is not shown to scale.CC-BY · From the PDF

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Citations

Cites

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Metadata

Authors
Star, B., Hansen, M. H., Skage, M., Bradbury, I. R., Godiksen, J. A., Kjesbu, O. S., & Jentoft, S.
Venue
STAR: Science & Technology of Archaeological Research
Publisher
Informa UK Limited
Published
2016-01
Volume
2
Issue
1
Pages
36-45
ISSN
2054-8923
DOI
10.1080/20548923.2016.1160594
Cited by
8
References
6
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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