Phi X 174

What is it?

1. a single-stranded DNA(ssDNA) virus that infects Escherichia coli
2. the first DNA-based genome to be sequenced in 1977
3. Well-defined, small(5,396bp), and diverse(45% GC, 55% AT) genome
4. fasta file download link:
1. PhiX_from_Illumina
2. PhiX_from_NCBI
5. Using it as a positive control in Illumina NGS

What are benefits of using PhiX control?

1. Calibration Control: can be run alone and serves as a calibration control for;
1. 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
   
   
2. Cross talk matrix generation
1. 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
2. https://www.slideshare.net/idtdna/unique-dualmatched-adapters-mitigate-index-hopping-between-ngs-samples
3. Phasing and Prephasing
1. During sequencing by synthesis, each DNA strand in a cluster extends by 1 base per cycle
2. 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)
3. For best results, use a PhiX spike-in as a control with any library that does not comprise a balanced base composition
4. High GC samples(≧ 60%) typically show higher phasing rates, and in this case a PhiX control is required


2. 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
   
   
3. Color balancing
1. For low diversity libraries, the PhiX Control v3 library provides balanced fluorescent signals at each cycle to improve the overall run quality
2. You can find why the nucleotide diversity is important in here

How to remove PhiX reads from the fastq