Quality reports

Version Compatibility

Reference examples tested with: FastQC 0.12+, MultiQC 1.21+, falco 1.2+, seqkit 2.5+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags
• Python: pip show <package> then help(module.function) to check signatures

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Quality Reports -- the traffic light is a hypothesis about WGS DNA, not a verdict

Generate per-file QC with FastQC/falco and aggregate the cohort with MultiQC, then READ THE PLOTS against the assay rather than trusting pass/warn/fail.

"Run quality control on FASTQ files" -> Compute per-base quality, composition, GC, duplication, and adapter profiles per file, then aggregate across samples to find outliers.

• CLI: fastqc -t 8 *.fastq.gz then multiqc .
• Long reads: NanoPlot --fastq reads.fastq.gz (FastQC assumes fixed-length short reads)

Scope: this skill OWNS raw-FASTQ QC reporting and interpretation, and carries the cross-cutting quality-score / chemistry / duplication concepts the rest of read-qc depends on. Remediation lives elsewhere -> read-qc/adapter-trimming, read-qc/quality-filtering, read-qc/fastp-workflow. Contamination -> read-qc/contamination-screening. Transcriptome QC on the BAM -> read-qc/rnaseq-qc. OUT OF SCOPE: any modification of the reads.

The Single Most Important Modern Insight

1. FastQC pass/warn/fail are heuristics calibrated to random whole-genome DNA, so they FALSE-FAIL on every other assay. RNA-seq fails per-base content (random-hexamer bias) and duplication (high-expression molecules); amplicon fails duplication and GC by design; bisulfite fails base content (C->T conversion); small-RNA fails length distribution; single-cell R1 fails everything (it is barcode+UMI, not biology). A red light is a HYPOTHESIS about a WGS library. For any other protocol, first ask "is this module expected to deviate for this chemistry?" before treating the red as a defect. Read the plot shape; the traffic light is calibration noise.

2. On 2-color chemistry (NextSeq, NovaSeq, MiniSeq) G is the ABSENCE of signal, so poly-G tails are called at HIGH quality and the quality plot will NOT flag them. When a cluster runs out of template, dark cycles read as a run of Gs with high confidence. Quality trimming alone does not remove them. They surface as a 3'-end RISE in G content (per-base sequence content) and a spurious high-GC spike, and they mis-map or manufacture false somatic variants if left in. The fix is a chemistry-aware poly-G trim (fastp auto-enables it from the instrument ID; cutadapt --nextseq-trim), not a quality cutoff. Read the per-base CONTENT plot on any 2-color run, not just the quality plot.

3. The duplication percentage is read-level and complexity-blind: it cannot tell a PCR jackpot from genuine high abundance. Identical reads from a highly expressed transcript, a targeted amplicon, or a ChIP/ATAC peak are counted as duplicates even though they are independent biological molecules. Duplication % is a function of BOTH library complexity AND sequencing depth (a good library sequenced deeply shows high duplication). It is a PROMPT to reason about library complexity (preseq), never an automatic "remove duplicates" -- and removing duplicates in non-UMI RNA-seq is actively wrong (read-qc/umi-processing, read-qc/rnaseq-qc).

Bonus trap: NovaSeq/NextSeq emit BINNED quality scores (RTA3 uses four values: 2, 12, 23, 37), so FastQC box plots look blocky/quantized. This is the instrument's quality table, NOT bad data and NOT something to fix. The bin edges are RTA-version-specific (NovaSeq X / RTA4 differs) -- never hard-code one bin set.

Tool Taxonomy

Tool	Role	Mechanism / when
FastQC	Per-file short-read QC (HTML + zip)	Java; the module set below; duplication/overrep from the first 100k distinct reads. The de-facto standard per-file report.
falco	Drop-in FastQC re-implementation (C++)	~3x faster, lower memory, same module names and MultiQC-compatible output. Use when FastQC throughput bottlenecks a large cohort.
MultiQC	Cross-sample aggregator (SCRAPER, not a re-analyzer)	Walks directories, regex-matches each tool's log/report, parses the numbers, builds one cohort report. The unit of review for multi-sample studies.
seqkit stats	Instant tabular FASTA/FASTQ numbers	seqkit stats -a: N50, Q20%, Q30%, GC%, length quartiles. For quick numbers and assembly/long-read contexts where FastQC is the wrong shape.
NanoPlot / NanoComp	Long-read (ONT/PacBio) QC	Read-length and quality distributions, yield, N50, length-vs-quality. The correct first pass for long reads; FastQC's fixed-length assumptions break there.

Decision Tree by Scenario

Scenario	Use	Why
Per-file Illumina short-read QC	FastQC (or falco)	Module-level diagnostics; read the plots by assay
Many samples / a study cohort	FastQC/falco then MultiQC	Outlier and batch detection is RELATIVE; only visible overlaid
Long reads (ONT/PacBio)	NanoPlot / NanoComp	FastQC is built for fixed-length short reads
Instant numbers, assembly input	seqkit stats -a	N50/Q20/Q30/GC in one line; no HTML overhead
Large cohort, FastQC too slow	falco then MultiQC	Same output, ~3x faster

Default when uncertain: FastQC on each file, then MultiQC over the run directory, and judge each sample against the cohort.

FastQC Modules -- thresholds and the expert read

Thresholds are FastQC's limits.txt defaults (calibrated to random WGS DNA). The expert read is what to conclude BEYOND the traffic light.

Module	Default warn / fail	Expert read
Per base sequence quality	warn LQ<10 or median<25; fail LQ<5 or median<20	3' decay is normal; blocky boxes on NovaSeq are binning; this plot will NOT reveal poly-G on 2-color
Per tile sequence quality	spatial deviation (no numeric)	A hot tile band across cycles = a localized flowcell problem (bubble, debris, edge); reason no MultiQC table replaces raw FastQC
Per sequence quality scores	distribution of per-read mean Q	A low-Q hump = a junk subpopulation to FILTER (not trim)
Per base sequence content	warn dev>10%; fail dev>20%	First ~12 bp skew = random-hexamer priming (Hansen 2010), expected for RNA-seq, do NOT trim it. A 3'-end skew is poly-G / adapter -- act on that
Per sequence GC content	warn dev>15%; fail dev>30%	SHAPE matters: bimodal/secondary peak = contamination; sharp spike = adapter dimer / overrepresented; a shifted single peak = wrong-GC reference assumption
Per base N content	warn N>5%; fail N>20%	Ns at a fixed position = a failed cycle; rising 3' Ns = dying clusters
Sequence length distribution	warn if lengths differ; fail if any length 0	WARNs trivially after trimming and on long reads -- ignore for those
Sequence duplication levels	warn if <70% would remain; fail if <50%	Read-level, complexity-blind (see insight 3); high = think complexity, not dedup
Overrepresented sequences	warn >0.1%; fail >1%	Most diagnostic module: it prints the sequence -- BLAST it (adapter dimer, rRNA, primer, poly-G)
Adapter content	warn k-mer>5%; fail >10%	A curve climbing toward 3' = read-through from short inserts; this panel IS an insert-size readout (route to adapter-trimming)
K-mer content	(deprecated, off by default)	Only appears in old reports; do not build guidance on it

Algorithm note (why duplication/overrep are estimates): FastQC tracks only the first 100,000 DISTINCT sequences, keys on the first 50 bp for reads >75 bp (so 3' errors do not fragment a duplicate family), counts by exact identity, and extrapolates the "% remaining if deduplicated" headline. It is a sample-based estimate, not a full-library dedup.

Duplication Taxonomy -- four causes, four actions

Class	Mechanism	Detected by	Action
Optical	One real cluster mis-segmented (non-patterned flowcell)	Same tile, pixel distance (Picard default 100)	Removable; spatially local artifact
ExAmp / patterned	One molecule seeds two nanowells (HiSeq X/4000, NovaSeq)	Spatially clustered, larger radius (Picard 2500 for patterned)	Removable; the reason patterned flowcells need the bigger pixel distance
PCR	Same fragment amplified and sequenced twice	Identical 5' coordinates post-alignment (+UMI if present)	Mark/remove for variant calling; NEVER coordinate-dedup amplicon (use UMIs)
Natural / biological	Independent identical molecules (high coverage, expressed genes, amplicon start)	Indistinguishable from PCR at read level without UMIs	KEEP -- removing biases quantification (do not dedup non-UMI RNA-seq)

The read-level duplication % FastQC reports cannot separate these. Use preseq (Daley & Smith 2013) to model the complexity curve and ask "how many NEW molecules would more sequencing buy?" -- that curve, not a single %, judges whether a library is exhausted or just deeply sequenced.

Quality scores and encoding

Phred Q = -10*log10(P_error): Q20 = 1% error, Q30 = 0.1%, Q40 = 0.01%. Q30 is the routine Illumina target; bulk Q40+ is uncommon on legacy chemistry (phasing, signal decay) and is a tell for re-binned or synthetic data on old runs, though XLEAP-SBS (NovaSeq X, NextSeq 2000) genuinely reaches Q40+. Modern data is universally Phred+33; any Phred+64 file (Illumina 1.3-1.7) feeds 31-too-high scores to a +33-assuming tool and passes garbage silently -- convert it (seqtk seq -Q64 -V). A quality byte below ASCII 64 (digits/punctuation) proves +33; detection tools sample reads to break ties.

MultiQC -- the cohort is the unit of review

MultiQC does NOT re-analyze data; it scrapes tool logs/reports (search_patterns.yaml), parses the numbers, and tabulates them per sample. Consequences: it is only as good as the files left on disk and the sample-name parsing (name collisions merge samples -- check multiqc_data/multiqc_sources.txt), and it reports whatever the upstream tool wrote (a wrong reference or wrong strandedness shows as a coherent-but-wrong table, not an error). Read the General Statistics table FIRST -- outliers jump out as a column anomaly -- then overlay per-base-quality / GC / duplication and ask whether the low-quality set maps to one lane / prep batch / operator. The batch effect caught here at QC is the one not chased for a month in the DE results.

# Per-file QC, then aggregate the run
fastqc -t 8 -o qc/raw/ raw_data/*.fastq.gz
multiqc qc/raw/ -o qc/multiqc/ -f

# Compare before vs after trimming in one report
fastqc -t 8 -o qc/trimmed/ trimmed/*.fastq.gz
multiqc qc/ -o qc/compare/ -f          # picks up both raw/ and trimmed/

# Long reads do not go through FastQC
NanoPlot --fastq ont_reads.fastq.gz -o qc/nanoplot/

Common Errors

Symptom	Cause	Solution
Every RNA-seq sample fails per-base content	Random-hexamer 5' bias (Hansen 2010)	Expected; do not trim the first bases
High-Q reads but a 3' G-content rise on NovaSeq	2-color poly-G (dark cycles = G)	Chemistry-aware poly-G trim (fastp / cutadapt --nextseq-trim), not -q
FastQC quality boxes look quantized/blocky	NovaSeq/NextSeq binned qualities (RTA3)	Expected; not a defect, do not "fix"
MultiQC merges two samples into one row	Over-aggressive name cleaning / collision	Check multiqc_sources.txt; use --fn_as_s_name or fix names
Duplication 60%, urge to dedup RNA-seq	Read-level dup is complexity-blind	Do not dedup non-UMI RNA-seq; assess complexity (preseq)
FastQC crashes / huge plot on long reads	Fixed-length short-read assumptions	Use NanoPlot / seqkit stats instead
FastQC module missing in MultiQC	The fastqc_data.txt was not on disk / wrong dir	Point MultiQC at the directory holding the zip/data files

References

de Sena Brandine G, Smith AD. 2019. Falco: high-speed FastQC emulation for quality control of sequencing data. F1000Research 8:1874. Ewing B, Hillier L, Wendl MC, Green P. 1998. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Research 8(3):175-185. Ewing B, Green P. 1998. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Research 8(3):186-194. Hansen KD, Brenner SE, Dudoit S. 2010. Biases in Illumina transcriptome sequencing caused by random hexamer priming. Nucleic Acids Research 38(12):e131. Daley T, Smith AD. 2013. Predicting the molecular complexity of sequencing libraries. Nature Methods 10(4):325-327. Ewels P, Magnusson M, Lundin S, Kaller M. 2016. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32(19):3047-3048. Shen W, Le S, Li Y, Hu F. 2016. SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation. PLoS ONE 11(10):e0163962. De Coster W, D'Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34(15):2666-2669.

Related Skills

read-qc/adapter-trimming - Remove read-through adapter flagged by the adapter-content panel read-qc/quality-filtering - Drop low-quality reads and trim ends read-qc/fastp-workflow - All-in-one QC + trim, including 2-color poly-G read-qc/contamination-screening - Resolve a bimodal-GC or unexpected overrepresented-sequence signal read-qc/rnaseq-qc - Transcriptome QC (strandedness, gene-body coverage) on the aligned BAM sequence-io/sequence-statistics - Programmatic per-file sequence summaries