Friday, 25 July 2014

PhiX Control - Phact or PhiXion?

Many of our users might have heard us talking about or seen a percentage of their reads aligning to the PhiX genome in the Multi Genome Alignment (see figure below). This is a result of Illumina's recommendation to use the PhiX genome control (see TechNote) for troubleshooting and quality control purposes. There are many features of PhiX that make it a good NGS control: it has a small 5386bp genome, it is a balanced genome (45% GC and 55% AT), and the library is 375bp average making it perfect for clustering and sequencing. The PhiX genome was the first genome to ever be sequenced.

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The Multi-Genome Alignment report
Why PhiX helps on the sequencer:
We use PhiX control in order to assess the quality of sequencing runs using Sequencing Analysis Viewer or SAV (see image below for an example). Illumina ship PhiX control at 10nM, which we then dilute, denature using our standard protocol and aliquot ready to use prior to clustering. As Illumina suggest, we spike in 1% of PhiX in lanes 1-7 and 5% in lane 8 in all our Hiseq runs, unless requested otherwise. We spike in 5% in our Miseq runs as we see more variable libraries being sequenced here. When checking run performance metrics in SAV, we check the cluster density, clusters passing filter (how many of the clusters are true clusters), error rate, phasing and pre-phasing and also the alignment rate to check the right amount of PhiX spiked in is aligning to the PhiX genome. These results can help us determine whether the problem is associated with the library or the machine, which is the reason we use PhiX to distinguish where the problem lies when a run or a lane doesn't perform well.

PhiX helps our troubleshooting: 
We check the run performance at every stage possible: we check after the clustering step to ensure the fluidics delivery is equal across all lanes, we check first base report to check the run looks good at the start of the sequencing and then we check the run metrics throughout the sequencing. When troubleshooting, we look at the same metrics as mentioned earlier, but in a lot more detail and also at many other metrics such as %base and the images to check that the library is balanced and to check the machine is behaving. When a run hasn't performed as expected and we cannot figure out the cause, we may also get Illumina involved and discuss the runs with them.

It can help get better results with funky libraries: There are many different library prep methods making it difficult to predict the performance of every sequencing run. Some methods such as Bisulfite, iClip, Bless, amplicons etc can produce "funky" libraries that might require the use of up to 50% PhiX.

The Genomics Core recommend using a higher percentage of phiX when:
  • You have a low diversity library (also under clustering can help here as well)
  • You have small amplicon pools
  • Bisulfite-seqeuncing, Blessd, amplicon sequencing

Here is the Illumina product code if you would like to order phiX:
PhiX control v3


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