1. Position specific effect of SNP on signal ratio from long oligonucleotide CGH microarrays; most single probe aberrations represent genuine genomic variants
Noyes HA1
Rennie K4
Hulme H2
Kemp SJ1
Hoyle DC3
Brass A2,4
Detection of copy number variations (CNV) on microarrays can be hampered by the presence of Single Nucleotide Polymorphisms (SNP). In order to be confident of calling genuine CNV rather than SNP, multiple contiguous probes are required to have non-zero log2 signal ratios. Consequently, only large CNV > ~5kb can be detected on typical CNV long-oligo arrays with probe densities 1 per 2kb. However the majority of CNV are probably <5kb (Nat Genet 2006, 38:82-85).
SNP data from the Perlegen 8 million SNP set and log2 signal ratios from ~300,000 long oligos were integrated in order to characterise the effect of SNP on log2 signal ratio and the effect of the position of the SNP within the probe. The maximum length of perfect match between probe and target appeared to be the dominant factor that affected hybridisation. The reduction in effective length of probe meant that single base changes could have a large effect on signal ratio and therefore be detectable on the long oligo arrays. Sequence differences were only expected to give high log2 signal ratios in our study design; therefore probes with low log2 signal ratios were potentially caused by CNV. Approximately 1000 probes with low log signal ratios were identified which were candidates for small CNV that would not have been identified by existing analysis approaches.
Most single probe aberrations appeared to be caused by genuine biological variants and were not due to experimental noise. Long-oligo CGH arrays can therefore provide more information than previously thought. The position specific effect of SNP will be useful for microarray design. A B
Large excess of high signal ratios when C57BL/6 used as common reference.
Agilent High density custom tiling array of 60 mer probes showing part of Mmu1. Note the much larger number of red probes (C57BL/6) with non zero log2 signal ratios than green (A/J) probes. In all experiments C57BL/6 DNA was used as a control and AJ, Balb/c or 129 was used as test times as many C57BL/6 probes had non zero log2 signal ratios as test strains in hybridisations with 3 different test DNA to two different array designs. Since the arrays were designed against the C57BL/6 genome sequence it is possible that many of the non-zero log2 signal ratios in C57BL/6 are caused by SNP in the test strains leading to poor probe binding.
1 School of Biological Sciences, BioSciences Building, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK
2 School of Computer Science, Kilburn Building, University of Manchester, Oxford Road, Manchester, M13 9PL, UK
3North West Institute of Bio- Health Informatics, School of Medicine, Stopford Building, Oxford Road, Manchester, M13 9PT, UK
4Faculty of Life Sciences, University of Manchester, Smith Building, Oxford Road, Manchester, M13 9PT, UK
Alternative models
Alternative models for the effect of SNP on hybridisation to target. In A the sequences between the two SNP act as independent probes and only the longest has a high enough melting temperature to bind at the annealing temperature of the reaction. In B the SNP cause small loops in the probe target duplex and has little effect on probe melting temperature.
Effect of SNP on log2 signal ratio
The global signal ratio was normalised to 0 but probes that contained a SNP in the Perlegen set had positive non zero log2 signal ratios and the ratio increased with number of SNP in the probe (r2 = 0.995). There was also a significant excess of probes with SNP amongst the probes that had a log2 signal ratio > 1 (p < 10-5).
Length of perfect match between probe and target determines risk of log2 signal ratio exceeding any given threshold
The strong association between the presence of SNP and positive log2 signal ratios suggested that model A in panel 1 was the dominant effect. If this is the case then the signal ratio would be related to the length of perfect match. The histogram shows the number of probes with log2 signal ratios > 0.3 and < 0.3 for each length of perfect match between 30 and 59 for probes with one SNP. The clear reduction in the number of probes with log2 signal ratio > 0.3 the nearer the SNP is to the end of the probe suggests that length of perfect match is an important component of probe target interaction.
Evidence for additional SNP not in Perlegen Set
Although there was a very large excess of SNP in the probes with high signal ratios only about 1/6th of the probes with high log signal ratios contained SNP within the Perlegen dataset.
In order to discover whether some of these probes might contain SNP not detected by Perlegen the 500bp region either side of each probe with a log2 signal ratio > 1 but no SNP was scanned for SNP. A significant excess of probes with log2 signal ratio > 1 had a SNP within 500bp compared with the same number of random probes, suggesting that many of the probes that did not contain a published SNP did in fact contain a SNP or other genomic variation.
Effect of Allele combinations on Signal Ratio
Although all SNP were associated with high log2 signal ratios some combinations of alleles appeared to be more destabalising than others. Pyrimidine to G mutations had the largest effect, these would lead to highly unstable GG or GA mismatches. The data also suggested that the orientation of the mis-matched bases on probe or target might be significant.
Possible additional small CNV
There were 1311 probes with log2 signal ratio < -1 in at least one strain and 519 in at least two strains. Since these are unlikely to have been caused by SNP they may have been caused by small CNV in the test strains. 32 of these probes were in regions that were found to be multicopy in C57BL/6 by BLAST search. Many of the remainder may be multicopy in the test strains but single copy in C57BL6.
ACKNOWLEDGEMENTS: We thank Leanne Wardlesworth for technical assistance and support from Tara Hall of Agilent. These studies were funded by the Wellcome Trust.