Base Calling
Base calling determines a base (A, C, G, or T) for every cluster of a given tile at a specific cycle. The iSeq 100 uses one-dye sequencing, which requires one dye and two images to encode data for the four bases.
Intensities extracted from one image and compared to a second image result in four distinct populations, each corresponding to a nucleotide. Base calling determines which population each cluster belongs to.
Visualization of Cluster Intensities
|
Base |
Dye in First Image |
Dye in Second Image |
Conclusion From Compared Images |
|---|---|---|---|
|
T |
On |
On |
Clusters that show intensity in both images are T bases. |
|
A |
On |
Off |
Clusters that show intensity in the first image only are A bases. |
|
C |
Off |
On |
Clusters that show intensity in the second image only are C bases. |
|
G |
Off |
Off |
Clusters that show intensity in neither image are G bases. |
Clusters Passing Filter
During the run, RTA2 filters raw data to remove reads that do not meet the data quality threshold. Overlapping and low-quality clusters are removed.
For one-dye sequencing, RTA2 uses a population-based system to determine the chastity (intensity purity measurement) of a base call. Clusters pass filter (PF) when no more than one base call in the first 25 cycles has a chastity below a fixed threshold.
PhiX alignment is performed at cycle 26 on a subset of tiles for clusters that passed filter. Clusters that do not pass filter are not base called and not aligned.
Index Reads
The process for base calling index reads differs from base calling sequencing reads. The first two cycles of an Index Read cannot start with two G bases, otherwise intensity is not generated. To ensure demultiplexing performance, intensity must be present in either of the first two cycles.
Make sure that at least one index adapter sequence in a library pool does not start with two G bases. Select balanced index adapter sequences so that signal is present in at least one image (preferably both images) for every cycle. The plate layout and sequences provided in Indexing Kit are designed to have the proper balance.
For more information on indexing and pooling, refer to the Index Adapter Pooling Guide (1000000041074) support page.
