Dear all,
Could you please explain the concept of "Unindexed clusters" as a result of Illumina sequencing? What leads to the appearance of such clusters? How do I interpret a high or low percentage of such clusters in my results? Also, what "filters" are appealed to clusters that are marked "PF" in, e.g., MiSeq output statistics?
Demultiplexing is done by providing a SampleSheet that contains a SampleID --> Index(s) association. Any index sequences that fail to match (even after allowing for 1 or 2 errors) result in those clusters being sent to "Undetermined" pool. You would generally want the percentage of clusters landing in undetermined pool to be as small a possible (generally 4-5% or less is norm).
Here is a page that should help you with all fields from a MiSeq statistics report. Chastity filter is used to decide if a cluster fails or passes (resulting in total PF%).
The Chastity filter works by calculating the ratio of the highest base intensity to the sum of the 1st and 2nd highest, anything less than 0.6 is filtered out. If a cluster was formed from a single-molecule then the chastity score will be 1; if it were formed from two molecules the signal would be equal and the chastity score will be 0.5.
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