Subject specific terms and wordings for NGS

When you start working with NGS, you will quickly notice that very specific wording is used for the different aspects. It is essential to understand these terms in order to be able to communicate or publish with colleagues in a reasonable way. Here we have tried to collect all these technical terms (based on Illumina machines) to save you this work.

Library preparation (library prep): Used method to prepare DNA or RNA for NGS.

Reverse Transcription: The conversion of RNA into cDNA when perfomring RNA-Seq.

Fragmentation: Fragmentation of the long DNA pieces into smaller fragments. Normally, this is done with the help of sonification. Please find more information here.

Size Selection: The selection of all fragments of a certain length.

Insert Size: Find an explanation here.

Fragments: Molecules to be sequenced.

Adapters: Oligonucleotides of a known sequence that are ligated to each end of fragments. They provide the primer sites used for sequencing the insert.

Adapter Ligation: Attachment of adapters to the 5' and 3' ends of each fragment.

Sequencing Library: A collection of DNA fragments of a particular size range with adapters ligated to each end.

Immobilization: The binding of the individual fragments on the flowcell. The adapter sequences bind to reverse complementary sequences on the flow cell.

Cluster Generation: Amplification of the individual immobilized molecules into clusters.

Bridge Amplification: Illumina specific, PCR-like amplification method.

Index/Barcode: Short sequences of 6-8 nucleotides used to identify/label individual samples when sequenced together in a single sequencing run. Indices are typically located within the adapter sequences.

Multiplexing: Mixing two or more different samples together such that they can be sequenced in a single sequencing run.

Target Enrichment: A method that allows to isolate specific genes or regions of interest prior to sequencing.

Hybrid-Capture Target Enrichment: Target enrichment by capturing genomic regions of interest by hybridization to target-specific biotinylated probes, which are then isolated by magnetic pulldown.

Amplicon Sequencing: Use of one or more pairs of PCR primers to increase the number of copies of genes or other regions of interest.

Gene Panels: Selected set of genes (or regions) that will be enriched.

Read: Nucleotide sequence of a part of the fragment returned by the sequencing machine.

Mate1 / Forward Read: Sequence part from the 5’ end of of the fragment.

Paired-End Sequencing: Special sequencing method from Illumina. After Mate1 is sequenced, all fragments in a cluster are flipped over and sequenced from the other side.

Mate2 / Reverse Read: Sequence part from the 3’ end of of the fragment. (only in paired-end sequencing)

Read Length: Number of sequencing cycles performed. All reads from one run (sequenced by Illumina machines) have exactly the same read length. Find more information here.

Coverage: Find more information here.

Sequencing Depth: Normally the amount of bases sequenced (i.e. 4 gigabases, 100 megabases). Sometimes it is used in a different context (i.e. amount of sequenced reads).

Library Complexity: High complexity means that there are very few identical sequences, whereas in a low complexity library there are very many copies (e.g. by PCR clones).

Strand-Specific RNA-Seq protocol: Find an explanation here.

Adapter Clipping: Find an explanation here or here.

Read Mapping: Finding the position of origin from which the read is most likely to come. Find a list of different mapping algorithms here.

Variant Calling: Detection of mutations (variants) using statistical algorithms.

SNP/SNV: Stands for Single Nucleotide Polymorphism and Single Nucleotide Variant.

InDel: Stands for Insertion or deletion.

CNV: Stands for Copy Number Variation

TMB: Stands for Tumor Mutational Burden

DE: Stands for Differentail Expression of mRNAs in different conditions.

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Last updated on May 11, 2019