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Whole genome amplification with SurePlex results in better copy number alteration detection using sequencing data compared to the MALBAC method.

Deleye L, De Coninck D, Christodoulou C, Sante T, Dheedene A, Heindryckx B, Van den Abbeel E, De Sutter P, Menten B, Deforce D, Van Nieuwerburgh F - Sci Rep (2015)

Bottom Line: Here, we compared the well-established SurePlex and MALBAC WGA for their ability to detect CNAs in MPS generated data and, in addition, compared PCR-free MPS library preparation with the standard enrichment PCR library preparation.Results showed that SurePlex amplification led to more uniformity across the genome, allowing for a better CNA detection with less false positives compared to MALBAC amplified samples.In general, the combination of SurePlex and MPS led to the same chromosomal profile compared to a reference arrayCGH from unamplified genomic DNA, underlining the large potential of MPS techniques in CNA detection from a limited number of DNA material.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.

ABSTRACT
Current whole genome amplification (WGA) methods lead to amplification bias resulting in over- and under-represented regions in the genome. Nevertheless, certain WGA methods, such as SurePlex and subsequent arrayCGH analysis, make it possible to detect copy number alterations (CNAs) at a 10 Mb resolution. A more uniform WGA combined with massive parallel sequencing (MPS), however, could allow detection at higher resolution and lower cost. Recently, MALBAC, a new WGA method, claims unparalleled performance. Here, we compared the well-established SurePlex and MALBAC WGA for their ability to detect CNAs in MPS generated data and, in addition, compared PCR-free MPS library preparation with the standard enrichment PCR library preparation. Results showed that SurePlex amplification led to more uniformity across the genome, allowing for a better CNA detection with less false positives compared to MALBAC amplified samples. An even more uniform coverage was observed in samples following a PCR-free library preparation. In general, the combination of SurePlex and MPS led to the same chromosomal profile compared to a reference arrayCGH from unamplified genomic DNA, underlining the large potential of MPS techniques in CNA detection from a limited number of DNA material.

No MeSH data available.


Scheme of read mapping after different amplification methods.MALBAC amplified samples have more uniquely mapped reads compared to SurePlex amplified samples. Samples were combined for the different starting amounts. Statistical analysis was performed with a one-way ANOVA followed by a Bonferroni t-test for multiple comparison. When comparing the percentages of non-uniquely mapped samples, the equal variance test failed. Here an ANOVA on ranks was performed followed by Dunn’s method for multiple comparison. #p < 0.001 and *p < 0.05.
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f4: Scheme of read mapping after different amplification methods.MALBAC amplified samples have more uniquely mapped reads compared to SurePlex amplified samples. Samples were combined for the different starting amounts. Statistical analysis was performed with a one-way ANOVA followed by a Bonferroni t-test for multiple comparison. When comparing the percentages of non-uniquely mapped samples, the equal variance test failed. Here an ANOVA on ranks was performed followed by Dunn’s method for multiple comparison. #p < 0.001 and *p < 0.05.

Mentions: When comparing the mapping results within each protocol from the samples with a different number of input cells, no significant differences were detected in the relative number of uniquely, non-uniquely and unmapped reads (p>0.05). A significantly higher percentage of reads, generated from the samples amplified with MALBAC, mapped uniquely compared to the percentage of uniquely mapped reads from samples amplified with SurePlex (82.8 ± 1.4% vs 76.1 ± 1.6%, respectively) (Fig. 4). This also holds true for the MALBAC and SurePlex amplified samples that were not enriched by PCR during library preparation (81.1 ± 2.4% vs 69.8 ± 2.3%, respectively). The percentage of uniquely mapped reads was significantly higher in the SurePlex amplified samples compared to the SurePlex amplified samples prepared without enrichment PCR. No significant difference was found for the MALBAC samples prepared with enrichment PCR versus the PCR-free MALBAC amplified samples.


Whole genome amplification with SurePlex results in better copy number alteration detection using sequencing data compared to the MALBAC method.

Deleye L, De Coninck D, Christodoulou C, Sante T, Dheedene A, Heindryckx B, Van den Abbeel E, De Sutter P, Menten B, Deforce D, Van Nieuwerburgh F - Sci Rep (2015)

Scheme of read mapping after different amplification methods.MALBAC amplified samples have more uniquely mapped reads compared to SurePlex amplified samples. Samples were combined for the different starting amounts. Statistical analysis was performed with a one-way ANOVA followed by a Bonferroni t-test for multiple comparison. When comparing the percentages of non-uniquely mapped samples, the equal variance test failed. Here an ANOVA on ranks was performed followed by Dunn’s method for multiple comparison. #p < 0.001 and *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4485032&req=5

f4: Scheme of read mapping after different amplification methods.MALBAC amplified samples have more uniquely mapped reads compared to SurePlex amplified samples. Samples were combined for the different starting amounts. Statistical analysis was performed with a one-way ANOVA followed by a Bonferroni t-test for multiple comparison. When comparing the percentages of non-uniquely mapped samples, the equal variance test failed. Here an ANOVA on ranks was performed followed by Dunn’s method for multiple comparison. #p < 0.001 and *p < 0.05.
Mentions: When comparing the mapping results within each protocol from the samples with a different number of input cells, no significant differences were detected in the relative number of uniquely, non-uniquely and unmapped reads (p>0.05). A significantly higher percentage of reads, generated from the samples amplified with MALBAC, mapped uniquely compared to the percentage of uniquely mapped reads from samples amplified with SurePlex (82.8 ± 1.4% vs 76.1 ± 1.6%, respectively) (Fig. 4). This also holds true for the MALBAC and SurePlex amplified samples that were not enriched by PCR during library preparation (81.1 ± 2.4% vs 69.8 ± 2.3%, respectively). The percentage of uniquely mapped reads was significantly higher in the SurePlex amplified samples compared to the SurePlex amplified samples prepared without enrichment PCR. No significant difference was found for the MALBAC samples prepared with enrichment PCR versus the PCR-free MALBAC amplified samples.

Bottom Line: Here, we compared the well-established SurePlex and MALBAC WGA for their ability to detect CNAs in MPS generated data and, in addition, compared PCR-free MPS library preparation with the standard enrichment PCR library preparation.Results showed that SurePlex amplification led to more uniformity across the genome, allowing for a better CNA detection with less false positives compared to MALBAC amplified samples.In general, the combination of SurePlex and MPS led to the same chromosomal profile compared to a reference arrayCGH from unamplified genomic DNA, underlining the large potential of MPS techniques in CNA detection from a limited number of DNA material.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.

ABSTRACT
Current whole genome amplification (WGA) methods lead to amplification bias resulting in over- and under-represented regions in the genome. Nevertheless, certain WGA methods, such as SurePlex and subsequent arrayCGH analysis, make it possible to detect copy number alterations (CNAs) at a 10 Mb resolution. A more uniform WGA combined with massive parallel sequencing (MPS), however, could allow detection at higher resolution and lower cost. Recently, MALBAC, a new WGA method, claims unparalleled performance. Here, we compared the well-established SurePlex and MALBAC WGA for their ability to detect CNAs in MPS generated data and, in addition, compared PCR-free MPS library preparation with the standard enrichment PCR library preparation. Results showed that SurePlex amplification led to more uniformity across the genome, allowing for a better CNA detection with less false positives compared to MALBAC amplified samples. An even more uniform coverage was observed in samples following a PCR-free library preparation. In general, the combination of SurePlex and MPS led to the same chromosomal profile compared to a reference arrayCGH from unamplified genomic DNA, underlining the large potential of MPS techniques in CNA detection from a limited number of DNA material.

No MeSH data available.