Read Quality Profile
Mean Phred, the fraction of the read above Q20 and Q30, the whole-read quality curve with the trimmed ends shaded, and the count of mixed peaks.
What it does
A Sanger read is not uniformly good. It starts badly while the polymerase and the capillary settle, runs clean through its middle, and decays as the fragments get long and poorly resolved. Every decision you make about the read — where to trim, whether to believe a variant, whether to re-run it — depends on knowing where on that curve you are looking.
This section shows the curve, and the three numbers that summarise it:
- Mean Phred over the whole read.
- ≥ Q20 / ≥ Q30, the percentage of bases at or above each threshold. Q20 is one expected error in 100 bases, Q30 one in 1,000.
- Mixed peaks (IUPAC), the number of positions called as an ambiguity code rather than a single base — the count that matters when you are looking for a heterozygous position.
The plot itself is a sliding-window mean of the per-base quality, with Q20 and Q30 drawn as reference rules so the read's good middle is a shape rather than a number, and the regions your trim discards shaded. The shading is on what is thrown away rather than what is kept, deliberately: the question you are asking of this plot is "am I cutting in the right place", so the eye should go to the cut.
When you'd use this
- Before trusting any base of a read. Look here first, then at the trace.
- When choosing where to trim. Set a trim in the section above and watch the shaded regions move against the curve — you are looking for the cut to land where the curve leaves the Q20 rule.
- When a variant call sits in a suspicious place. A mismatch inside a stretch below Q20 is a read problem until proven otherwise; the same mismatch at Q45 in the middle of the read is real.
- When deciding whether a read is worth keeping at all. A read whose ≥Q20 fraction is small has no good middle, and a re-run is cheaper than the argument about what it shows.
- When you expect a heterozygote. The mixed-peak count is the fast answer to "did the base caller see any ambiguity at all in this read".
What this computes
- Mean Phred over the read, from the base caller's own values as read from the file rather than recomputed — the same numbers your sequencing provider quotes. Q = −10 log₁₀ P(wrong base), so Q20 is 1 error in 100, Q30 is 1 in 1,000 and Q40 is 1 in 10,000
- The fraction of bases at or above each of two thresholds
- A windowed quality profile, with the window scaled to the read at read length ÷ 40, clamped between 5 and 40 bases, stepping by half a window so neighbouring points overlap. A fixed window would be wrong at both ends of the range — 20 bases is noise on a 200-base read and over-smoothing on a 1,200-base one — and the window size in use is printed under the plot beside the read length, so the smoothing is never hidden. Each point sits at the centre of the bases it summarises, not at the window's left edge
- The current kept range, in 1-based inclusive coordinates
- The mixed-peak count
What it refuses to invent: a read from a format carrying no per-base quality gets no curve. The section says the trace carries none and shows the mixed-peak count alone.
Where it lives
The Quality & peaks section of the Chromatogram panel, reporting on the active track — the read whose tab is in front, so switching tabs re-points the section rather than pinning it to the read it was opened from. Its header carries a detach control that floats the whole section — the numbers, the plot and the mixed-peak count together, not just the chart.
Inputs and outputs
In: the active trace's per-base quality values, as read from the file. AB1 and SCF files carry them; a read from a format that does not is not given an invented curve — the section says the trace carries no per-base quality and shows the mixed-peak count alone.
Out: mean Phred, the two threshold fractions, the windowed profile, the current kept range in 1-based inclusive coordinates, and the mixed-peak count.
Controls
| Control | What it changes |
|---|---|
| Active tab | Which read the section describes. |
| Trim start / end (Trim section) | Moves the shaded discarded regions on the plot and the "keeping a–b" readout under it. |
| Detach | Floats the section beside the canvas. |
The plot's own window size is not a knob, and that is deliberate — see below.
The science
Phred quality is Q = −10 log₁₀ P(wrong base), so Q20 is a 1-in-100 error rate, Q30 is 1-in-1,000 and Q40 is 1-in-10,000. The values are the base caller's own, taken from the trace file rather than recomputed, so they are the same numbers your sequencing provider quotes.
The window scales with the read, at read length ÷ 40, clamped between 5 and 40 bases, and it steps by half a window so neighbouring points overlap. A fixed window would be wrong at both ends of the range: 20 bases is noise on a 200-base read and over-smoothing on a 1,200-base one. The window size in use is printed under the plot alongside the read length, so the smoothing is never hidden.
Each point is plotted at the centre of its window, over the bases it summarises, rather than at the window's left edge.
A window with no quality values is a gap in the line, not a zero. Q0 is a real and meaningful score — "definitely wrong" — so substituting it for "unknown" would draw a confident cliff that is not in the data. The line breaks instead.
Shared with other tools: Read quality compares how every tool that does this chooses its method.
Limitations
- The curve is smoothed, so a single catastrophic base does not show as a spike. For per-base quality read the trace itself, where each base carries its own value.
- Quality is the base caller's opinion, not a measurement of the DNA. A clean-looking trace with a mixed template can score well and still be wrong about which molecule you sequenced.
- Mean Phred over the whole read is a weak summary — it mixes the bad start, the good middle and the decayed end into one number, which is exactly why the plot is here. Do not report it as a quality metric for the useful part of the read; trim first, then read the numbers.
- The mixed-peak count is a count, not a call. It says how many positions carry an ambiguity code; whether any of them is a real heterozygous site is a question for the trace and the alignment.
Related
In this group: Edit annotation · Enzyme · Insert sequence · Merge features · Manage enzymes · Dot plot · Dot plot grid · Melt curve · Amplification curves · Population genetics · RNA-seq · Site avoidance heat map · Protein coverage · Reference-gene stability · Digital PCR · Codon usage matrix · Pairwise distance distribution · Nearest-neighbor distances · GC content distribution · Base composition per cycle · Pairwise distance grid · Per-cycle channel occupancy · Grammar assembly plan · Gibson assembly plan · Assembly plan build order · Golden Gate assembly plan · Guide track · Primer binding track · Hydropathy profile · Charge vs pH · Amino-acid composition · Hydrophobic moment · Optimization profiles · Digest gel · Virtual gel · Initiation-window fold · Antibody domain map · Electropherogram · Circuit netlist · Circuit transfer functions · Circuit construct · Construction graph · Protocol · Read Alignment · Raw Signal · Verify vs Reference · Chromosomal microarray · Variant calls · Small molecule
Protocol
A construct's recorded build read as numbered sentences, followed by steps you write on the sequence open now, each answered directly under it.
Read Alignment
The analyse operations on an open set of reads: screen for vector, align two reads head to head, open all of them as a multiple alignment, and phase a heterozygote into its two alleles.