Question: Unindexed clusters: Illumina
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gravatar for mariafirulevabio
9 months ago by
mariafirulevabio40 wrote:

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?

ADD COMMENTlink modified 9 months ago by genomax80k • written 9 months ago by mariafirulevabio40
2
gravatar for genomax
9 months ago by
genomax80k
United States
genomax80k wrote:

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.

ADD COMMENTlink modified 9 months ago • written 9 months ago by genomax80k
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