Illumina and TotalSeq™

Illumina’s sequencing and array technologies are fueling groundbreaking advancements in life science research, translational and consumer genomics, and molecular diagnostics. Illumina sequencing platforms have been adopted by leading institutions around the globe, and are the primary platforms used to sequence single-cell and bulk RNA/DNA sequencing libraries.

All TotalSeq antibodies contain a capture sequence, antibody barcode, and a partial Read 2 PCR handle that is compatible with Illumina sequencing platforms. The PCR handle acts as a priming location to append the full-length Read 2 sequence and both the i7 sample index and P7 sequencing adaptor. Following capture of the Antibody Derived Tag (ADT) or Hashtag Oligo (HTO), the Read 1, i5 sample index, and P5 adaptor are added during the subsequent library construction (method used dependent upon platform). Following completion of the single-cell workflow, full-length dual index libraries are created containing the necessary adaptors and indices to be sequenced on Illumina sequencing platforms.

TotalSeq™ Antibody Structure

Generic ADT/HTO Final Sequencing Library

*structure of final ibrary may vary between single cell platforms

Sequencing Library Components	Function
P5 and P7 adapters	Sequences that allow the library to bind and generate clusters on Illumina flow cell.
Read 1 and Read 2	Sequencing primer binding sites to initiate sequencing.
i5 and i7 Sample Index	Sample identifiers that allow multiplexing/pooling of multiple samples in a single sequencing run or flow cell lane.
Cell barcode	Identifies which cell this sequencing is associated with.
Unique Molecular Identifiers (UMI)	Identifies the specific molecule the library is derived from; used to address the presence of PCR duplicates generated during library amplification.
Capture sequence	Sequence used to capture ADT/HTO oligonucleotide following single cell partioning.
Antibody barcode	Sequence used to identify the antibody.
Sample Index	The barcode used to identify which pool or sample the sequencing library belongs to.

Illumina Sequencing Platforms

Illumina has several compatible sequencing platforms for single-cell and bulk sequencing. Below is a list of the most commonly used systems. For more information, visit Illumina sequencing platforms.

Learn More About Illumina NGS

Illumina Support

Bulk Epitope and Nucleic Acid Sequencing (BEN-Seq) with TotalSeq-A™

BioLegend and Illumina have collaborated to develop the BEN-Seq multiomic assay that enables the simultaneous profiling of cell surface proteins and RNA expression at the bulk level. Explore our page to learn more about the utility and development of BEN-Seq.

Discover BEN-Seq

BioLegend Recommended Sequencing Depth (reads/cell)

TotalSeq™-A, B, and C Libraries

Library Type	Minimum Sequencing Depth (reads/cell)
3’ and 5’ Gene Expression Library	Follow platform recommended guidance
Cell Surface Protein Library <100 ADT panel	5,000
Cell Surface Protein Library ≥100 ADT panel	10,000
Cell Hashing Libraries*	500

*Note: If using TotalSeq-A-independent cDNA, ADT and HTO libraries are made allowing the user to attribute reads/cell to each library. This is not possible for TotalSeq-B and -C ADT and HTO libraries as they are constructed in the same cDNA pool and standard reads/cell should be followed based on the total number of antibodies used.

TotalSeq™-D Protein Libraries

Mission Bio recommends an average coverage of 750x per cell per antibody. This represents the total number before filtering to allow for adequate read depth post-filtering and cell calling. The total number of reads required for one sample can be calculated as follows:

Total reads with 10-AOC panel (example): Number of expected cells x number of amplicons x recommended coverage

V2 5,000 x 10 x 750 = 37.5 M

V3 11,000 x 10 x 750 = 75 M

Sequencing Run Parameters, Library Loading, and Pooling

Refer to the platform-specific user guides for guidance on run parameters, library loading, and pooling. For additional information regarding denaturing and diluting libraries, refer to the corresponding documentation for the sequencer you are using.

If using TotalSeq-A, you can find run parameter and pooling guidance in our TotalSeq-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3.1 (Dual Index) Protocol.

Illumina Knowledge contains additional information, FAQs, troubleshooting articles, and reference material for Illumina products and workflows, covering all Illumina microarrays and every stage of sequencing.

Illumina Data Sets

BioLegend Cell Hashing/TotalSeq libraries sequencing runs now available on Illumina® BaseSpace™ Sequence Hub

BioLegend BEN-Seq libraries sequencing runs now available on Illumina® BaseSpace™ Sequence Hub