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.

1. 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.

1. 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

Decision Tree by Scenario

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.

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

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 (searchpatterns.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 multiqcdata/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

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