Infinium MethylationEPIC Array Scanning on NextSeq
NextSeq Control Software (NCS) v4 enables low/mid-throughput MethylationEPIC BeadChip scanning on the NextSeq 550 system. While NCS v4 is compatible for use on NextSeq 500 and 550 systems, only NextSeq 550 systems are capable of BeadChip scanning.
For more information, see the Infinium MethylationEPIC BeadChip support page and the Infinium MethylationEPIC Kit product page on the Illumina website.
Array Scanning on NextSeq versus iScan
The iScan is specialized for scanning all Infinium beadchips. While the iScan device can be outfitted with an autoloader to facilitate high-throughput scanning, it can’t sequence nucleic acid samples. The NextSeq 550 offers both low/mid-throughput CytoSNP and MethylationEPIC beadchip scanning, without the use of an autoloader, and also offers high-throughput sequencing. If you plan to focus on large sample volume studies (> 5000 samples/year) relying on arrays, the iScan is a better choice. If you plan studies that do not require a high volume (up to 5000 samples/year) and would like to incorporate sequencing into your studies as well, the NextSeq 550 is ideal.
The NextSeq 550 supports the same Infinium workflow and analysis pipeline for MethylationEPIC analysis as the iScan. NextSeq 550 supports all MethylationEPIC samples that the iScan can process (fresh samples, FFPE, etc.).
Comparing the Data
Intra-platform variance between NextSeq 550 instruments is negligible. We also saw a strong correlation between results obtained on two iScan devices and three NextSeq 550 instruments. R-square and β values show a strong correlation within the same instrument type as well as between iScan and NextSeq 550.
Scanning results compared to iScan device #1
| Cell Lines | Fresh Frozen | FFPE | ||||
|---|---|---|---|---|---|---|
| R-square | % delta 20 | R-square | % delta 20 | R-square | % delta 20 | |
| iScan #2 | 0.998 | 0.003 | 0.989 | 0.011 | 0.963 | 0.036 |
| NextSeq #1 | 0.996 | 0.003 | 0.982 | 0.019 | 0.937 | 0.062 |
| NextSeq #2 | 0.996 | 0.003 | 0.983 | 0.017 | 0.947 | 0.051 |
| NextSeq #3 | 0.995 | 0.004 | 0.979 | 0.021 | 0.94 | 0.058 |
Design of the optical systems in each instrument is different, which is manifested in a lower measured intensity on NextSeq 550. Normalization of the intensities results in precise calculation of methylation changes on the NextSeq 550 as well as the iScan. Our data shows that, regardless of intensity, key precision specifications of MethylationEPIC are maintained. Differences in β values between samples of 0.2 are measurable with 99% confidence. Comparison of β and intensity values for NextSeq 550 and iScan shows that β values remain comparable for the two instrument types.
Scanning results of several NextSeq instruments compared to an iScan device
While our data show that NextSeq 550 and iScan provide comparable results, there can be experimental or equipment-specific nuances in different environments that confound comparisons between an iScan device and a NextSeq 550 instrument. In these situations, we recommend carrying out application specific validations.
Normalizing the Data
To compare data generated on NextSeq 550 to data from an iScan device, we recommend using standard normalization algorithms that convert signal intensity to beta values. Normalization should remove the effect of signal intensity differences prior to analysis. The same guidance applies if you want to compare data between systems.
If the NextSeq system has been regularly serviced, the age of the instrument has no impact on normalization methods or data quality.
There are public repositories that contain idat files for experiments carried out with MethylationEPIC beadchips using the iScan. The same normalization techniques used to process NextSeq 550 data can be used with publicly available raw data collected with the iScan. Even with signal normalization, Illumina urges that any comparative results with historical iScan data be validated on the NextSeq 550 instrument.
Additional Resources
- Infinium MethylationEPIC Sample Sheet
- Infinium MethylationEPIC Product Files
- Infinium MethylationEPIC Demo Data Set (NextSeq 550 System)
- Infinium Methylation Support Documentation
- Specifications
Technical Bulletins
- The correct storage temperature for Infinium BeadChips is 2-8°C
- DNA strand designations
- Infinium HD Methylation assay workflow for fresh/frozen versus FFPE samples
- Infinium Methylation450K manifest column headings
- Which bisulfite conversion kit should I use for the Infinium Methylation arrays?
- Improve Infinium data with these updated XStain glass back plate care recommendations
- Recommendations for Infinium Array XC4 reagent use and storage