I only worked with the Ion Torrent till now, but now I have to use the MiSeq. I´m first of all a little bit confused by the terms used: is the read number equal to the number of clusters you can have on your flow cell? Also what is the typical loss in reads that you can observe? I´m trying to calculate how many samples we can analyze and be sure to have a good coverage.
Thanks in advance!
Hi,
This is a relatively involved question because it depends on a number of factors. Which sequencing kit are you using (i.e. 2x150)? Optimal cluster density will correlate roughly to the total bases you should get from a run. We generally shoot for around 950K mm2 and generate about 4.5-5 Gb of data >90% Q30 (2x150). What's important to think about is how your libraries are generated--for a basic prep without any type of capture your PCR duplicate rate will be low, but some capture techniques have higher duplicate rates that will affect how you many samples you might wish to run. Perhaps some more information regarding what you are sequencing, how it's prepped, and how many total bases you expect to sequence would be helpful to give you a ballpark estimate of what you might wish to try.
More simply, the total number of clusters is equal to the total number of reads in a single read mode run. However some reads are rejected by the Illumina filter to give you the "PF read number". The specs say that you should have a minimum of 80% of the original reads passing filtering. If yo do not overload the flow cell, you will typically get > 90% passing filter rate (PF %).
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology