Genpathway Specifications for Illumina Sequencing of ChIP DNA Libraries
- Length of sequence reads: > 35 bases.
Rationale: 35-mer sequence reads by Illumina sequencing is now standard for ChIP-Seq. This length is desirable to maximize mapping to unique sequence locations in the genome.
- Number of sequence reads obtained per channel: > 8 million and preferably > 10 million.
Rationale: Specified number of sequences is required for adequate coverage across the genome and quantitation of IP’ed sequences in the original ChIP DNA. Sequencing of fewer reads per channel may also be an indication of lower quality of the cluster formation and/or sequencing steps.
- Ability to work with Genpathway’s libraries submitted as follows:
a. Total of 0.2-1 ug DNA in 10 ul
b. Concentration of 20-100 ng/ul e. Concentration to use for cluster formation based on Q-PCR titration carried out by Genpathway using Illumina protocol
c. Concentration measured by Nanodrop
d. Photo of gel demonstrating range of DNA sizes
Rationale: Genpathway’s libraries are generated according to Illumina protocols and will be submitted at typical concentrations and total amounts. Titration is needed for optimal cluster formation, and is carried out using an Illumina protocol in place of a preliminary cluster formation run on an Illumina instrument. Based on this titration, the concentration to use stated by Genpathway must be followed in order to maximize the number of clusters obtained prior to sequencing. Use of another concentration could lead to generation and/or imaging of lower cluster numbers. The Nanodrop concentration provided by Genpathway must also be used, e.g., rather than a Bioanalyzer concentration, based on information from Illumina. The gel photo is provided to show the range of library DNA lengths and can take the place of a Bioanalyzer run.
- Use of Eland or Eland Extended for alignment.
Rationale: Alignment must be carried out using a proven alignment tool, of which Eland has been shown to be the fastest. Eland Extended includes multiple map locations (up to 10) and mismatches (up to 2), both of which are denoted on the sequence align list.
- Output files formatted according to Eland export files.
Columns required include: Chromosome, genomic location of sequence start, genomic location of sequence end, and strand. Standard quality scores must also be included.
Rationale: Genpathway will incorporate the output files into its comprehensive data analysis software, and uses output files in this format.
- Minimum of 10 successful ChIP sequencing runs to date.
Rationale: The sequencing laboratory must have successful experience in carrying out Illumina sequencing on ChIP DNA libraries.
© 2010 Genpathway, Inc.
Chromatin and Methylated DNA Immunoprecipitation
"ChIP" Products & Services
"ChIP" Products & Services
