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 idea seems to have been first implemented in an iCLIP protocol (König et al.

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/

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 on the way the barcodes are added to the library. The gist of it is: For barcodes added to Illumina libraries

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 the artifacts "index hopping".  Please see the index hopping information from Illumina here and in this video

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 data but single-end data are also usable.

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. Yields can vary depending on library type.

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 out of essential reagents - in most cases the PCR primers will be exhausted.  If primers are no longer available the PCR products will anneal to each other  (the sequencing adapter sequence will be the by far most common sequences available).

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.

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 contain a file with the de-multiplexing metrics and a file listing md5 checksums  for all the FASTQ files.