I did sequencing run using PhiX control in Illumina's NextSeq 500 and found out the %PF only 0.4% yet the cluster density reached 267K/mm2. The reagent I used was fresh (not expired yet), it was the high-output reagent for 300 cycles. I did paired-end sequencing. I wonder if anyone have already experienced this and how to figure out the problems because I'm not sure if overclustering is the main reason since the cluster density is quite ok.
As you say, the cluster density is well within the normal range. Can you provide a little more information? Do you mean that you did a run with only PhiX DNA included in the library, or that you sequenced your own experimental samples with PhiX DNA included as a small percentage of the total pooled library (as the typical Illumina protocol recommends)? Also, if you're using BaseSpace can you post a screenshot of the "Run & Lane Metrics" tab from the run in question? Is it the "CLUSTER PF (%)" column that is at 0.4%? If instead it is either the "ALIGNED (%)" or "ERROR RATE (%)" column, those would be pretty typical values.
Hi Jesse
Yes I did a run with only PhiX DNA, so it's supposed to produce a decent result since PhiX function as a control. I used SAV to analyse the QC result and it mentioned that "cluster PF(%)" was the one with value 0.4% thus my data output was very small, only 0.4Gb. I'm curious if there are other factors out of "overclustering" that made the clusters can't pass the chastity filters. Could be the reagent or the optic the source of the problem?
Thanks for your recommendations!
Hi Juli,
It does seem like there's a problem with ambiguous base calling early in the run that's preventing clusters from passing the filter. According to Illumina, "During cycles 1–25 of Read 1, the chastity filter removes the least reliable clusters from the image extraction results. Clusters “pass filter” if no more than 1 base call has a chastity value below 0.6 in the first 25 cycles. Chastity is defined as the ratio of the brightest base intensity divided by the sum of the brightest and the second brightest base intensities." I'm not sure what would produce low chastity values other than overlapping clusters with different bases generating mixed signals. I guess it's possible that the clusters are not distributed evenly over the flow cell surface, instead occurring in clumps that are too close together (you might be able to get a sense of this from the raw image files. How do the other columns in the SAV output look (e.g. error rate, Q score, phasing/prephasing %, % aligned)? Those values might give you a sense of whether there could be a reagent or optics issue.
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