Pooling samples

Introduction 

Multiplexing (pooling) several samples into a single sequencing run can significantly reduce costs. To allow for multiplexing, each sample is tagged with a unique barcode during amplification. The barcode will then be used to demultiplex the reads after sequencing.

How to pool

For iRepertoire’s arm-PCR amplification technology, we provide two product options (depending on the size of your project) that will require pooling. For small-scale projects, our iR-Profile kits contain everything you need to amplify and individually barcode 10 samples. For larger-scale projects, we recommend purchasing multiple iRepertoire Bulk Primer Kits with different barcodes. In both cases, each sample is amplified in its own reaction, and then samples are pooled together prior to sequencing submission. Our data analysis software will identify and differentiate the samples based on the barcodes used.

For more information on iRepertoire’s products, see our products page.

As an example, if you want to study the human TCR beta repertoire of 200 samples using your own Illumina HiSeq, you should purchase iRepertoire catalog items HTBIvc-01-P to HTBIvc-20-P, which will provide you with short-read bulk primer reagents for barcodes 1-20. Then, you can use each of the 20 kits to amplify the first 20 samples. After arm-PCR, the 20 libraries can be pooled for one single HiSeq lane. The same 20 kits can be used again for the next 20 samples for the second HiSeq lane, etc. Currently, the Illumina HiSeq instrument can run two flow-cells in the same instrument run, totaling 16 lanes. So, using 20 barcodes for one lane, all 200 samples can be sequenced in a single HiSeq run.

For iRepertoire’s dam-PCR technology, the barcoding strategy is similar, except that dam-PCR must be performed as a service or through our automated iR-Flex platform. When performed as a service, iRepertoire’s technicians handle the barcoding strategy. For the iR-Flex platform, which can also be used for arm-PCR, the automated cassettes each have a single barcode.

For more information about arm-PCR and damPCR, see the Learning Center article about iRepertoire’s amplification technology.

To explore our data analysis software, visit our Bioinformatics page.

For more information about our iR-Flex platform, see our automation page.

Note on sequencing depth/coverage

Because sequencing coverage is determined by the number of reads per sample, and each sequencing run produces a defined number of reads, multiplexing samples will decrease the coverage of each individual sample. Sequencing depth will thus need to be considered when planning a multiplexed sequencing project.

The number of samples pooled will depend on your experimental needs. For instance, if the samples are disease-related and you expect a clonal expansion of just a few clones, you may be able to pool up to 60 samples in a lane (depending on barcode availability) and still achieve your goal.  

For other projects, you may require deeper sequencing of a single sample. If this is the case, we recommend amplifying the same sample with 10 different barcodes on an Illumina HiSeq lane, or 4 different barcodes on the Illumina MiSeq Flow Cell, and treating them as different samples. 

For more information about sequencing coverage, see our Learning Center article about choosing read depth