What is it?
- a single-stranded DNA(ssDNA) virus that infects Escherichia coli
- the first DNA-based genome to be sequenced in 1977
- Well-defined, small(5,396bp), and diverse(45% GC, 55% AT) genome
- fasta file download link:
- Using it as a positive control in Illumina NGS
What are benefits of using PhiX control?
- Calibration Control: can be run alone and serves as a calibration control for;
- Cluster generation: can be used as a positive control in the clustering process
Platform Mode/Reagents Optimal Raw Cluster Density HiSeq High Output, TruSeq v3 750-850 K/mm² High Output, HiSeq v4
(required upgrade)950-1050 K/mm² Rapid v2 850-1,000 K/mm² MiSeq v2 1,000-1,200 K/mm² v3 1,200-1,400 K/mm² MiniSeq Mid and High Output 170-220 K/mm² NextSeq Mid and High Output, v2 170-220 K/mm² [table 1] Cluster density guidelines for Illumina sequencing platforms - Cross talk matrix generation
- During an illumina sequencing run, the cross-talk due to spectral overlap between the 4 fluorescently labeled nucleotides is calculated during template generation in cycle 1-5
- https://www.slideshare.net/idtdna/unique-dualmatched-adapters-mitigate-index-hopping-between-ngs-samples
- Phasing and Prephasing
- During sequencing by synthesis, each DNA strand in a cluster extends by 1 base per cycle
- A small proportion of strands may become out of phase with the current cycle, either falling a base behind(phasing) or jumping a base ahead(prephasing)
- For best results, use a PhiX spike-in as a control with any library that does not comprise a balanced base composition
- High GC samples(≧ 60%) typically show higher phasing rates, and in this case a PhiX control is required
- Run quality monitor: due to its small size and balanced nucleotide composition, it's an ideal in-run control (typically with >= 1% spike-in) for run quality monitoring
Platform PhiX Aligned(%) iSeq 100 minimum 5% MiniSeq 10~50% MiSeq
(MCS 2.2 or higher)minimum 5% NextSeq 10~50% HiSeq 2500
(HCS 2.2.38 or higher)minimum 10% HiSeq 3000/4000
(HCS 3.3.76 or lower)10~50% HiSeq 3000/4000
(HCS 3.4.0 or higher)5~20% NovaSeq minimum 10% [table 2] PhiX Control v3 library Illumina recommends spiking in when running low diversity libraries - Color balancing
- For low diversity libraries, the PhiX Control v3 library provides balanced fluorescent signals at each cycle to improve the overall run quality
- You can find why the nucleotide diversity is important in here
How to remove PhiX reads from the fastq
