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What are the major differences between Illumina & Roche 454 and how do they apply to your technology?
 

Next-generation sequencing (NGS) of the immune repertoire allows for the sequencing of millions of V-regions (or V(D)J sequences) in parallel. However, many of the NGS platforms are limited by the read-length of the output sequencing reads. iRepertoire offers several reagent systems to take advantage of the various output lengths, and ALL reagent systems cover the CDR3 portion. Please refer to the table below for a summary of the differences between the NGS platforms (as it applies to iRepertoire’s products and services).

 

 

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.

 

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