Sample Submission Guidelines1992 Bisulfite Treatment for Methylation Samples
Commonly referred to as BS conversion, it involves treating methylated samples with bisulfite (abbreviated as "BS"), where "conversion" refers to the process by which unmethylated cytosines (C) are converted to uracils (U) and eventually to thymines (T) through PCR amplification, while methylated cytosines (5mC) are preserved.
2003 Pyrosequencing
Pyrosequencing is primarily used for validating methylation sites. Essentially, it combines bisulfite modification with PCR amplification to operate a pyrosequencing machine. Notably, BS pyrosequencing has become the gold standard for methylation validation due to its high accuracy.
2005 MeDIP-seq, RRBS, NGS
MeDIP-seq, short for Methylated DNA Immunoprecipitation Sequencing, relies on antibody-based enrichment to selectively gather methylated genomic fragments for sequencing. MBD-seq (Methylated DNA Binding Domain Sequencing) can be considered its counterpart, focusing on regions where methylated proteins bind to DNA.
RRBS, or Reduced Representation Bisulfite Sequencing, relies on methylation-sensitive restriction enzyme digestion. By using enzymes like MspI for genome digestion, RRBS enriches CpG sites. The coverage of CpG sites depends on the "CCGG" restriction sites, and it dynamically changes with fragment size selection. Although extensions such as double-enzyme RRBS and laser capture microdissection RRBS have been explored, the fundamental principle remains laboratory enzymatic digestion, limiting RRBS applications.
Furthermore, it’s worth noting that from this year onwards, next-generation sequencing (NGS) technology emerged, leading to the integration of methylation detection with NGS, resulting in numerous new technologies and applications.
Services you may interested in
2007 27k Chip
This was the first methylation detection chip introduced by Illumina, known as Infinium Human Methylation 27 Beadchip. It contains approximately 27,578 probe pairs targeting CpG sites, hence referred to as the "27k" chip. At that time, it covered a considerable number of methylated sites, and traces of its use can still be found in the TCGA database. While Illumina is often associated with high-throughput sequencing today, its initial product was actually a gene chip.
2009 WGBS
WGBS, or Whole-Genome Bisulfite Sequencing, involves WGS combined with BS conversion. Theoretically applicable to any species, it requires a substantial amount of input DNA, typically at the microgram level. Improved methods have reduced this requirement to as little as 1ng for detection. However, its performance is still not entirely satisfactory. The vigorous treatment of DNA during bisulfite conversion results in significant information loss. Moreover, it is costly, being the most expensive among known high-throughput methylation detection techniques.
2011 450k Chip
As NGS technology was applied to methylation detection, the number of known methylated sites on the human DNA dramatically increased. Consequently, Illumina upgraded the 27k chip to the Infinium Human Methylation 450 BeadChip, capable of detecting 482,421 CpG sites and 3,091 non-CpG cytosines. Commonly referred to as the "450k" chip, it has long been the preferred choice in the methylation detection market, overshadowing similar chip products.
2012 oxBS-seq, TAB-seq, and 5hmC
These are two methods for detecting 5hmC. OxBS-seq, short for Oxidative Bisulfite Sequencing, involves oxidative bisulfite treatment. TAB-seq, or TET-Assisted Bisulfite Sequencing, employs the TET-assisted sodium bisulfite sequencing method.
2013 SureSelect Methylation Capture Technology
In 2013, Agilent introduced their methylation capture product, the Agilent SureSelect MethylSeq Kit. It utilizes a method of capture followed by bisulfite conversion to obtain information on methylated sites. Subsequent sequencing via NGS allows for detection. Although its detection throughput is slightly lower than WGBS, it is significantly higher than the 450k (and later mentioned 850k) arrays. Moreover, unlike RRBS, it doesn’t exhibit positional bias, making it a highly cost-effective methylation detection product.
2016 850k (EPIC) Chip
Towards the end of this year, Illumina upgraded its chip product, introducing the Infinium HumanMethylationEPIC BeadChip, an upgrade to the Methylation Chip 3.0. Capable of detecting approximately 853,307 CpG sites, it’s dubbed the "850k" chip. This chip has been extensively used in scientific research, testing tens of thousands of samples annually.
2019 TAPS Technology and Customized Capture
Traditionally, methylation detection relied on BS conversion, which, despite its flaws, outperformed other sample processing techniques. However, in February 2019, a new contender emerged: TAPS (TET-Assisted Pyridine Borane Sequencing). Unlike BS sequencing, TAPS does not require sodium bisulfite and allows for direct DNA methylation sequencing of target sequences. It is a less destructive and more efficient single-base resolution DNA methylation sequencing method. TAPS adopts a strategy that differs from BS conversion, allowing for the direct detection of 5mC and 5hmC without affecting unmodified cytosines. This method has been commercialized by startup companies for developing cancer liquid biopsy screening products.
Also in this year, Twist and Qiagen each introduced their customized methylation capture kits. They both employ a strategy of BS conversion followed by capture, requiring lower input DNA amounts, typically in the tens of nanograms, making them more suitable for cfDNA blood testing in clinical settings. Grail, a hot company in cancer early screening, uses Twist’s methylation custom technology.
2023 935k (EPIC) Chip
Illumina launched the Infinium Methylation EPIC v2.0 (Illumina 935K Methylation Chip) in 2023. This product is an upgrade to the Illumina Methylation EPIC v1.0 (Illumina 850K Methylation Chip). With over 935,000 CpG sites, the 935K Methylation Chip maximizes compatibility with the 850K Methylation Chip. It has been evaluated based on the 850K Methylation Chip, removing and replacing poorly performing probes.
2023 Infinium Methylation Screening Array
The Infinium Methylation Screening Array focuses on 270K unique methylation sites associated with common traits, disease phenotypes, environmental exposures, aging, and more. Abbreviated as MSA270K, it achieves a highly accurate single-chip detection throughput increase of 48 samples, six times that of the Infinium Methylation EPIC v2.0. This array offers higher throughput and lower cost.