What are UMIs and why are they used in high-throughput sequencing?

UMI is an acronym for Unique Molecular Identifier.  UMIs are complex indices added to sequencing libraries before any PCR amplification steps, enabling the accurate bioinformatic identification of PCR duplicates. UMIs are also known as “Molecular Barcodes” or “Random Barcodes”.  The

Posted in

Where can I find a tutorial on Illumina and NGS sequencing?

Illumina has posted a Beginners Guide on their technology at: https://www.illumina.com/science/technology/next-generation-sequencing/beginners.html Please also see our information at: https://dnatech.genomecenter.ucdavis.edu/illumina-high-throughput-sequencing/

Posted in

How should I submit the barcode sequence information? In which direction will they be sequenced?

Depending on sequencer and in case of the HiSeq 4000 even depending on run type (single-end or paired-end) Illumina uses different approaches to sequence the indices.   Please find detailed information here: indexed-sequencing-overview-guide-15057455-04-Illumina-pages1to8 The correct orientation of the barcode sequence fuehrer depends

Posted in

Which indexing scheme should I use for Illumina sequencing to prevent index hopping? (UDI adapter)

Illumina sequencers using the patterned flowcell technology (HiSeq 4000, NextSeq 2000, HiSeq X Ten, NovaSeq, iSeq) can show an increased rate of barcode switching events.  These artifacts are enabled by the exclusion amplification chemistry used on these sequencers; IIlumina calls

Posted in

How should I sequence ATAC-seq libraries?

It is generally recommended to sequence 50 million or more reads/library-molecules  per ATAC-seq sample for open chromatin detection and differential analysis (Buenrostro et al. 2015) and 200 million reads for TF footprinting (Yan et al. 2020).  Preferred are paired-end sequencing

Posted in

What read numbers/yields can I expect from Illumina sequencing?

The Illumina specifications are based on the Illumina PhiX control library. Better or similar yields can be expected for other high complexity libraries (e.g. genomic, RNA-seq libraries) if they are within the recommended insert size ranges and do not average extreme GC-contents.

Posted in

My libraries show peaks larger than expected. Can I still sequence these PCR-bubbles?

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.  These PCR artifacts do occur in cases the PCR reactions run

Posted in

How should I prepare and sequence samples for ChIP-seq?

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

Posted in

In which form will I receive the data?

All sequencing data will be available for secure download via our SLIMS server. Illumina sequencing data will be delivered as compressed FASTQ files. By default the data will be de-multiplexed (e.g. split according to sample).  Each SLIMS directory will further

Posted in

Can I use custom sequencing primers? What melting temperatures should these have?

Please note that custom sequencing primers are not supported by Illumina — the company will not replace sequencing kits on run failures. Nevertheless, custom sequencing primers can enable some unique assays. It is certainly worth exploring if assays can’t be

Posted in