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Pooling Samples


One catalog item (such HBHI-M-01-P) includes enough reagents to produce 10 libraries (all of which will have the same barcode). However, to best utilize the Illumina or Roche 454 sequencing capacities, libraries generated from several samples, each with a different barcode, can be pooled together in one single HiSeq lane, MiSeq Flow Cell, or Roche 454 plate. Ultimately, this reduces the per sample sequencing cost.


How to Pool


To study multiple samples in the same sequencing run, you need to purchase multiple iRepertoire primer kits with different barcodes, amplify the samples separately, pool the PCR products together before submitting the pooled library for sequencing. Our data analysis software will identify and differentiate the samples based on the barcodes used during arm- PCR.



For example, if you want to study the human TCR beta repertoire of 200 samples using the Illumina HiSeq, you should purchase iRepertoire catalog items “HTBIvc-01-P” to “HTBIvc-20-P.” Then, you can use each of the 20 kits to amplify the first 20 samples. After arm-PCR, the 20 PCR products 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.


A Note on Sequencing Depth


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 achieve your goal. However, 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, 4 different barcodes on the Illumina MiSeq Flow Cell, and 2 on the Roche 454 platform and treating them as different samples. The use of different barcodes facilitates downstream data processing and can help in the discovery of infrequent CDR3 sequences. 


Coverage and Read Estimation


As for coverage, we recommend 5 reads for each cell so that theoretically every cell will be sequenced according to the Poisson model. For instance, if your sample contains about 500,000 T cells, we recommend you allocate about 2.5 million reads for this sample. For an estimation of the read output (estimated read output or ERO), the average number of estimated sequencing reads after SMART filtering is divided by the number of samples pooled (S). Please refer to the calculations below (due to variations in sequencing run parameters beyond the control of the operator, estimates may be higher or lower than actual output). 


  •   Illumina HiSeq 2000 Lane:  100 x 10^6 / S = ERO

  •   Illumina MiSeq Flow Cell: 10 x 10^6 / S = ERO

  •   Roche 454 plate: 1 x 10^6 / S = ERO


For example, an Illumina HiSeq2000 lane with 40 samples pooled has an ERO of 2.5 million reads per sample.