Methylation

The following can be modified in order to optimize hybrid capture:

• Hybridization time – Start with 2 hours as recommended and modify between 30 min to 4 hours.
• Fast Wash Buffer 1 Temperature – Start with 65°C, adjust between 63-66°C:

Expected final library yield is between 50-75ng/ul. The yield will depend on the quality of the starting input.

CpG Methylated pUC19 DNA and Unmethylated Lambda DNA are included in the kit. These controls can be used to determine the efficiency of enzymatic conversion during library preparation.

NOTE: Internal controls should not be included in the capture workflow unless control-specific probes are added to the enrichment panel.

See table: 

We recommend a minimum of 10-20 ng of high-quality DNA and maximum of 200 ng.

Yes, multiplexing up to an 8-plex is supported. We recommend using 200 ng of library for a single plex and 1500 ng (or 187.5 ng each) for an 8-plex capture.

There is no detriment to any hybrid selection metrics when using this reagent. It reduces off-target in a variable fashion depending on custom methylation panel target regions and methylation states of the input genomic DNA.  It can potentially decrease 50% in off-target in some custom panels.

Twist only supports Mechanical Fragmentation for this application when building libraries to undergo methylation conversion.

200 to 300 bp

No. Hybrid Capture must be performed with the Twist Fast Hybridization System.

350 to 450 bp

The following can be modified in order to optimize hybrid capture:

• Hybridization time – Start with 2 hours as recommended and modify between 30 min to 4 hours.
• Fast Wash Buffer 1 Temperature – Start with 65°C, adjust between 63-66°C:

Expected final library yield is between 50-75ng/ul. The yield will depend on the quality of the starting input.

CpG Methylated pUC19 DNA and Unmethylated Lambda DNA are included in the kit. These controls can be used to determine the efficiency of enzymatic conversion during library preparation.

NOTE: Internal controls should not be included in the capture workflow unless control-specific probes are added to the enrichment panel.

See table: 

We recommend a minimum of 10-20 ng of high-quality DNA and maximum of 200 ng.

Yes, multiplexing up to an 8-plex is supported. We recommend using 200 ng of library for a single plex and 1500 ng (or 187.5 ng each) for an 8-plex capture.

There is no detriment to any hybrid selection metrics when using this reagent. It reduces off-target in a variable fashion depending on custom methylation panel target regions and methylation states of the input genomic DNA.  It can potentially decrease 50% in off-target in some custom panels.

Twist only supports Mechanical Fragmentation for this application when building libraries to undergo methylation conversion.

200 to 300 bp

No. Hybrid Capture must be performed with the Twist Fast Hybridization System.

350 to 450 bp