Takara SMARTer Human BCR data now available on new 600 cycles kit on the NextSeq 2000

The human adaptive immune system is responsible for the specific recognition and elimination of foreign antigens originating from infection and disease. The molecular recognition of antigens is mediated through the enormous diversity of antibody (B cell receptors) and T cell receptors.

The immune repertoire is defined as the collection of all T cell receptors (TCRs) and B cell receptors (BCRs; also known as immunoglobulins) within an individual. Sequencing the expressed T and B cell receptor genes in the immune repertoire provides a detailed picture of a person’s health. Recent launch of the new 600 cycles kit on the NextSeq2000 is a very compelling solution for immune repertoire applications which benefit from longer reads. We are showing here sequencing data generated with Takara SMARTer Human BCR IgG IgM H/K/L Profiling libraries.

Library preparation

Takara SMARTer Human BCR IgG IgM H/K/L Profiling libraries (catalog number 634467) pairs 5' RACE with NGS technology to provide a sensitive, accurate, and optimized approach to BCR profiling from RNA input samples. Please note that Takara recently launched SMART-Seq Human BCR (with UMIs), which improves upon the existing chemistry through incorporation of UDI sample barcodes and captures the complete V(D)J variable regions of all human BCR heavy (IgG/M/D/A/E) and light (IgK/L) chains.

Takara libraries were prepared using total RNA extracted from 50mL whole blood from single healthy donor provided through AllCells. To better understand the effects of input RNA quantity on required sequencing depth and clonotype saturation 5x different libraries were prepared with 25, 50, 100, 500, 1000 ng RNA inputs, respectively. Simultaneously 6x 100ng replicates and 4x 1,000ng replicates were generated to better understand technical reproducibility of the assay

Accessing sequencing run

Takara libraries were sequenced on the NextSeq1000/2000 P2 300M Reagents (600 Cycles) catalog number 20075295. To view Takara SMARTer Human BCR IgG IgM H/K/L Profiling libraries run on NextSeq2000 600 cycles kit, please access the BaseSpace^TM Sequence Hub and import the sequencing run using the link provided below.

Run link: https://basespace.illumina.com/s/N9pY62l0yTOp

These demo data are free to use on the BaseSpace Sequence Hub platform (see our previous post - https://developer.illumina.com/news-updates/demo-data-on-illumina-basespace-sequence-hub)

Evaluation of the sequencing run

To understand how to evaluate your sequencing run, see our previous blogpost "Does my sequencing run look good?"

Takara SMARTer Human BCR IgG IgM H/K/L Profiling libraries were sequenced with a read length of 301 + 8 + 8 +301 on the NextSeq1000/2000 P2 300M Reagents (600Cycles), catalog number 20075295. Due to the lower base diversity present in specific regions of full-length antibodies, 30% PhiX (v/v) was spiked in the pool of libraries to increase base diversity. These libraries also contain a UMI barcode making it possible to both quantitively determine transcript abundance as well as correct reads derived from PCR errors or duplication and/or sequencing errors, thus ensuring more accurate and reliable results.

Figure: sequencing data by cycle showing the data by cycle for Q30 (top) and percentage base (bottom).

Analysis

Project link contains the analyzed data: https://basespace.illumina.com/s/xk4fkqnfPQ2O

FASTQ generated on the NextSeq 2000 were analyzed on the Takara Cogent NGS Immune Profiler Software. The Takara Cogent NGS Immune Profiler summarizes major statistics collected during processing and merges the results into multiple comma-separated value (CSV) files that can be viewed in a spreadsheet program. Included in the summary outputs include detailed information regarding read QC metrics, mapping statistics, and clonotype details. Summary metrics can be found in the BaseSpace project link for each sample.

Additional resources:

To answer your questions regarding preparation and sequencing of Takara immune repertoire libraries, contact our experts at Takara (technical_support@takarabio.com). For questions about your sequencing run, contact Illumina (techsupport@illumina.com).

If you need further information on Takara immune repertoire technology, please refer to: https://www.takarabio.com/products/next-generation-sequencing/immune-profiling/human-repertoire

For Research Use Only. Not for use in diagnostic procedures.