Illumina sequencing libraries are usually generated with Y-adapters. These are partly single-stranded and partly double stranded. A PCR-free library will thus still contain partly single-stranded regions. These single-stranded regions can lead to several types of Bioanalyzer artifacts:
PCR amplified sequencing libraries frequently display library molecules seemingly about twice the excepted size or even bigger. In most cases, this phenomenon is caused by over-amplification of the libraries.
If we prepare the sequencing libraries we require ChIP-seq DNA samples to be submitted after reversal of the cross-linking. Ideally, the fragment lengths should be between 100 and 300 bp, and preferably under 500 bp. The former will result in the tightest peaks.
For ChIP-seq it is common to start with DNA samples with concentrations too low to measure.
If you have access to fluorometric DNA quantification and a Bioanalyzer (or equivalent), library pooling is not difficult. We offer the pooling of sequencing libraries for a small fee.
We have currently 96 indices are available and can pool 96 RNA-seq or genomic sequencing libraries. Bioo Scientific offers NEXTflex barocde sets allowing the pooling of up to 384 libraries. If you are planning to use homebrew versions of indices please consult with us first, as reduced complexity from incorrectly designed indices may cause failures when sequencing your sample.
Quality and quantity of DNA and RNA is critical for high quality sequencing output. Please make sure your DNA is not degraded and is free of RNA contamination. RNA samples should always be assessed on the bioanalyzer for the absence of gDNA contamination (can be removed with DNaseI treatment followed by a column clean-up; e.g. Zymo "RNA Clean and Concentrator") and degradation. Preferentially determine the concentrations of your DNA and RNA samples using fluorometry (e.g. with a Qubit or plate reader). The sample purity should be assessed by spectrophotometry (e.g.
