When designing RNA-seq or ChIP-seq experiments, it is very important to avoid technical replicates and pseudo-biological replicates as they will lead to spurious results (e.g. spurious differential gene expression data; DGE data in case of RNA-seq). Creating pseudo-biological replicates occurs…

In case the library preparation did not generate sufficient library material required to load a sequencer, Illumina libraries can be amplified with a universal PCR protocol. While the amplification can rescue experiments, it is worth considering on a per-project basis…

Ampure XP/SPRI bead “upper cut” protocol to remove double-stranded DNA fragments over 670 bases: Bead-based size selections are the preferred method as they can be applied in a high-throughput fashion to your samples.  Please note: Bead-based size selection cannot carry…

RNA-seq experiments should best be carried out with samples of consistent RNA integrity and input amounts.  However, some RNA-seq samples can be so limited and irreplaceable that experiments have to be carried out with less than the recommended input amounts.…

Is PCR-free library preparation still advantageous? In general, the original concerns about library PCR amplification (presented in papers from 2008) are no longer very relevant.  This is due to the use of modern polymerases that are designed for complex samples…

The HiSeq 4000 sequencer is the most demanding Illumina sequencer with regards to library insert sizes.  Nevertheless, the majority of existing Illumina sequencing libraries can be sequenced as is on the HiSeq 4000: The libraries should not have any or no visible…

Primer and adapter-dimer contamination in sequencing libraries can lead to serious problems like barcode switching (also called barcode hopping).  Thus, these short molecules should be removed from the libraries as soon as traces of them become visible on the Bioanalyzer or…

3’Tag-Seq is a protocol to generate low-cost and low-noise gene expression profiling data.   The protocol is also known as  TagSeq, 3’Tag RNA-Seq, Digital RNA-seq, Quant-Seq (please note that most of these names have also been used for a variety of other protocols previously). In contrast…