HI ,
I wanna do multiplexed sequencing validation from this paper' Technical Validation of a Next-Generation
Sequencing Assay for Detecting Actionable Mutations in Patients with Gastrointestinal Cancer'.
"Multiplexed Sequencing Validation :
We used the Illumina Sequencing Coverage Calculator to estimate the number of samples that could be multiplexed in our assay based on the total sequencing capacity per run of the MiSEQ and our desired coverage of 700 to 1000.This estimate of the number of multiplexed samples per run was 10 to 15.
To validate multiplex sequencing, one run of four FFPE specimens was used. The analysis of positions with nonreference allele(s) in one FFPE sample but absent in the other samples allowed us to estimate on-instrument cross-talk frequency of 0.01% to 0.04% (Table 5). The total error rate, as ½T5 estimated by fraction of any nonreference bases called at such positions in the other samples, was 0.03% to 0.23%, which is far less than the limit of detection of the GI panel test."
table 5 :
I wanna know how to do this part . and can you give me some detailed steps ? thank you !
