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Qualtiy Control Expert Q&A

Legend:

  • FP = false positive result
  • END = no solution, this problem is unsolvable

RNA-seq

  1. high peak at low bp in the electropherogram (intensity mV per Size bp)
  • source: documentation (PDF)
  • possible reason(s): contamination e.g. adapter dimers (adapter+adapter, no DNA)
  • solution/measure: increase starting material / trim /exclude by size
  1. combine replicates from multiple batches without any replicates within each batch
  • source: conceptional
  • possible reason(s): it is not possible to distinguish between the biological and the technical variance -> bad design
  • solution/measure: END
  1. enrichment of wrong population (e.g. polyA-enrichment to compare to rRNAs)
  • source: conceptional
  • possible reason(s): it is not possible to distinguish between the biological and the technical variance -> bad design
  • solution/measure: exclude rRNAs

QC

  1. low base call quality at 3'-end
  • source: fastqc, fastq input (sam/bam are missing Phred)
  • possible reason(s): general effect esp. for older data, could be hint for 3'-adapters
  • solution/measure: check trim again
  1. overall low base call quality
  • source: fastqc, fastq input (sam/bam are missing Phred)
  • possible reason(s): universally very good reads + wrong phred-type detection (FP)
  • solution/measure: check encoding detected by fastqc, could be a FP
  1. red spots in per tile sequence quality
  • source: fastqc, Illumina input
  • possible reason(s): damaged flow cell
  • solution/measure: exclude reads from these cells, buy new flow cell
  1. high per base sequence content
  • source: fastqc, Illumina input
  • possible reason(s): adapter sequence still present
  • solution/measure: trim adapters
  1. at the 5' end high per base sequence content
  • source: fastqc, Illumina input
  • possible reason(s): TSS reads -> start of read = start of gene = bias distr. (FP)
  • solution/measure: -
  1. C is missing in high per base sequence content
  • source: fastqc, Illumina input
  • possible reason(s): sodium bisulphite treated = C->T (FP)
  1. overall high high per base sequence content
  • source: fastqc, Illumina input
  • possible reason(s): contamination e.g. adapter dimers
  1. 2/more peaks in per sequence GC content
  • source: fastqc, Illumina input
  • possible reason(s): multiple unrelated species present
  • solution/measure: remove contaminated species
  1. 2/more peaks in per sequence GC content
  • source: fastqc, Illumina input
  • possible reason(s): other contamination, e.g. adapter dimers
  • solution/measure: adapter dimers => trim
  1. high percentage of overrepresented sequences
  • source: fastqc, Illumina input
  • possible reason(s): contamination present in reads
  • solution/measure: blast most abudant reads, can be adapter sequences too
  1. high percentage of overrepresented sequences
  • source: fastqc, Illumina input
  • possible reason(s): overrepresented sequence present in data e.g. rRNAs (FP)
  1. high percentage of duplicated sequences
  • source: fastqc, Illumina input
  • possible reason(s): low complexity
  • solution/measure: little starting material / heavy PCR
  1. high percentage of duplicated sequences
  • source: fastqc, Illumina input
  • possible reason(s): constrained library (only reads starting at TSS) (FP)
  • solution/measure: You can use random barcoding to distigush between biol. and tech. replicates if needed

Trimming

  1. no reads are trimmed although adapters are present
  • source: trim-galore, Illumina input
  • possible reason(s): hard coded adapter sequences, ...
  • solution/measure: adjust parameters
  1. trimmed reads although no adapters are present
  • source: trim-galore, Illumina input
  • possible reason(s): false positive result of TG
  • solution/measure: skip trim-galore

Mapping

  1. high number of unmapped reads
  • source: Mapping tool / output, Illumina input
  • possible reason(s): wrong target genome/transcriptome
  • solution/measure: -
  1. high number of unmapped reads
  • source: Mapping tool / output, Illumina input
  • possible reason(s): adapter sequence still present
  • solution/measure: go back to trimming

Post-Mapping

  1. different conditions correlate very good
  • source: correlation matrix, Illumina input
  • possible reason(s): low potential for DEGs present
  • solution/measure: END
  1. within replicate correlation is bad
  • source: correlation matrix, Illumina input
  • possible reason(s): e.g. contamination with HVG like rRNAs
  • solution/measure: if only a small sub-population is confounding -> remove
  1. replicates do not cluster
  • source: PCA, Illumina input
  • possible reason(s): e.g. biol. and tech. replicates are mixed up
  • solution/measure: END-RESTART
  1. replicates do not cluster
  • source: PCA, negative control study
  • possible reason(s): negative control study (FP)
  1. two experiment of 2 conditions (ctrl, KO) cluster in the wrong area
  • source: PCA, negative control study
  • possible reason(s): misslabeling
  • solution/measure: check design matrix, documentation, ...
  1. no clear separation between conditions (ctrl, KO)
  • source: PCA, negative control study
  • possible reason(s): e.g. biol. and tech. replicates are mixed up
  • solution/measure: END-RESTART

DEA

  1. dispersion-plot: gene estimation does not follow red fit
  • source: DESeq2, negative control study
  • possible reason(s): model does not represent data
  • solution/measure: Deseq is not applicable
  1. dispersion-plot: high fit dispersion for high mean count
  • source: DESeq2, negative control study
  • possible reason(s): low number of replication + high variability
  • solution/measure: careful with reported DEGs
  1. almost all genes are DE
  • source: DESeq2, negative control study
  • possible reason(s): e.g. biol. and tech. replicates are mixed up
  • solution/measure: END-RESTART
  1. almost no gene is DE
  • source: DESeq2, negative control study
  • possible reason(s): negative control study (FP)

Visualize

  1. using CPM to visualize DEGs
  • source: Plot, negative control study
  • possible reason(s): mixed up within- and between-sample normalization
  • solution/measure: use correct normalization
  1. use raw Ratios to visualize DEGs effect size
  • source: Plot, negative control study
  • possible reason(s): humans are bad with ratios (0.01 = almost 0 and 100 is just large but not the largest bar ever)
  • solution/measure: use any log transformation (e.g. log10: 0.01 => -2, 100 => +2)

Single cell

Quality check

  1. peak at left/right side in gene or reads per cell histogram or log10-cummulative-number of reads per cell id
  • source: BD rhapsody pipeline, negative control study
  • possible reason(s): left=cell fragments, right=multiplets present
  • solution/measure: remove with cut-offs
  1. inflated UMI counts
  • source: BD rhapsody pipeline, negative control study
  • possible reason(s): using raw UMI counts
  • solution/measure: use DBEC/RSEC UMI counts

Dim. reduction

  1. poor PCA/UMAP/tSNE embedding
  • source: Dim. reduction embedding, negative control study
  • possible reason(s): using e.g. only one assay of count data for embedding
  • solution/measure: for multimodal data use a WNN approach (combining both assays)
  1. poor PCA/UMAP/tSNE embedding
  • source: Dim. reduction embedding, negative control study
  • possible reason(s): use raw data for tSNE/UMAP
  • solution/measure: use a significant portion of PC from the PCA as input for tSNE/UMAP
  1. "The first two principle components were used to perform a tSNE" https://doi.org/10.1126/science.aag3009
  • source: Dim. reduction embedding, negative control study
  • possible reason(s): use only 2 PC from the PCA for the tSNE/UMAP projection
  • solution/measure: use a significant portion of PC from the PCA as input for tSNE/UMAP

Find subpopulations

  1. cluster form based for a specific batch index
  • source: Dim. reduction embedding + clustering, negative control study
  • possible reason(s): batch effect
  • solution/measure: correct for batch effect (e.g. integrate using seurat)

DEA

  1. many DEGs
  • source: seurat/deseq, negative control study
  • possible reason(s): DEG between very small sub-populations
  • solution/measure: use a population size cutoff or state the number
  1. some genes look like dates (1-Mar,...)
  • source: seurat/deseq, negative control study
  • possible reason(s): some genes can be interpreted as dates when using excel for data handling https://doi.org/10.1126/science.aah4573
  • solution/measure: never ever use excel or at least make sure that cell type is not "AUTO"

Further resources

References