iPlant – Intermediate workshop

UCDavis, 9/22


Harold Pimentel, PhD student in CS at UC Berkeley

“Algorithms for RNAseq – from raw reads to differential expression analysis”



Not all reads are created equal! Number reads proportional to length of transcript, normalize to correct for length bias


Very fast overview of RNAseq workflows. New RNAseq workflow faster. Typically align reads (e.g. bowtie or splice-aware tophat). Genomic vs. transcriptomic alignment? Genomic alignment is easier to visualize. Tools are similar, similar results.

Pseudoalignment with Kallisto.

At the end of the day, what you care about is counting matches with transcripts. Can do analysis on laptop in <10 min, similar accuracy to existing methods.


Take k-mers of every single transcript, walk along path. Wherever they differ, you create a new path. In dB graph, different splice sites diverge along path of graph, sometimes they have same path but some don’t. Follow k-mers along graph, intersection of k-mers can give upper bound on compatibility of prior kmers in path. First 3 k-mers have exact same information, 2nd only have a few in common, same with the next. Method always assumes

Performance speed >500x faster than existing tools, comparable accuracy.

Two schools of thought for RNAseq differential expression: transcript abundance estimation vs. counts

Issues with raw counts (Figure 1). Counts do not take into account length, not well posed: Isoform A (1000) vs. Isoform B (500) vs. union of A and B (1500)

FPKM union <= FPKM true

Sum of fractions vs. fractions of sums

How wrong is gene counting? Take a closer look at cuffdiff2 approach (paper).

Latent allocation problem. Maximum likelihood estimate number of reads of one vs. num

L. Pachter “Models for transcript quantification from RNA-Seq” arXiv, 2011. Review of models, all transcript quantification methods boil down to inference algorithm. Then you have abundance estimation output for sailfish, kallisto. For example, proportionally assign A, fragment B which is compatible only with red therefore unique mapping. Re-estimate maximum likelihood estimate, iterate and prove likelihood and will be non-decreasing every step every good as last step. Under some circumstances guaranteed local maximum and under some global maximum.

Results are highly dependent on particular transcriptome, mapping step parameters, and what is considered to be compatible. What if missing annotation, get partial mapping with one complete mapping to another isoform. Take this on case by case basis, not really a blanket statement can be made about every scenario of analysis. A few papers have tried to answer, but when you’re missing 15-20% of transcriptome, depending on what your’e missing ( what you’re missing is probably not highly expressed).

  • Simple t-test won’t work because not normal distribution, negative binomial (NB2) (DESeq2, edgeR)
  • assume log(counts) = normal (sleuth, limma)


Between sample normalization, spike-ins are not to always be trusted (from published studies). Naive vs. not-so naive normalization: TMM, DESeq median count/geom. mean across samples.

Dealing with small sample sizes. Estimators have variance, this variance is large in small samples, large variance on your variance! Prior shrinking weights from weight towards some prior. eBayes estimator to define what weights are going to be. Key principle. Increasing power by pooling all information together. If only one estimate, not enough information. Many estimates across many different genes, exploit this.

Multiple hypothesis testing problem. (Null true but rejected=type I error) If we do this thousands of times, the number of false-positives blows up, even if there isn’t real discovery.

Sleuth has bootstrap, multinomial model to resample data and get new transcript abundance estimates. Essentially, in silico technical replicates. Take sample, bootstrap variance has high correlation. Assume that noise is Poisson distributed, sometimes not true. Especially transcript abundance estimated variance.

Try Snakemake for creating reproducible workflows!


  1. don’t compare raw units without normalization
  2. every choice you make in a pipeline affects downstream
  3. RNAseq should be tool for guidance

(References on slides at end.)

Kallisto and sleuth walkthrough: https://github.com/pimentel/bears_iplant

About Lisa Johnson

PhD candidate at UC Davis in Molecular, Cellular, and Integrative Physiology
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