1. No reference available
Design experiment
Prepare libraries
Conduct sequencing
Produce FASTQ files
Pre- process data
No reference available
Assembly of genome sequence
Reference available
Map reads to reference genome or transcriptome
Assembly of transcriptome sequence
RNA transcripts
DNA modifications
Protein Interactions
Variant Discovery

2. Map reads to reference genome or transcriptome
Design experiment
Prepare libraries
Conduct sequencing
Produce FASTQ files
Pre- process data
Auer & Doerge, 2010
Genetics 185:405-16
DNA sequences A
DNA sequences B
Fu et al, 2014,
PNAS 111:1891-6
Map reads to reference genome or transcriptome

3. Experimental design
Genetics 185(2):405-16, 2010
Here seven treatments are applied to each of three biological replicates , and each sample is sequenced in a separate lane
Are nuisance effects confounded with treatment effects?

4. Experimental design
Yes – all samples from each rep are on the same flowcell.
Flowcell effects are confounded with replicate effects, and
lane effects are confounded with treatments, because T1 is always in lane 1, T2 in lane 2, and so forth.
Genetics 185(2):405-16, 2010

5. Experimental design
Auer & Doerge,
Genetics 185:405,
2010

6. Experimental design
Same experiment: seven treatments applied to each of three biological replicates , but samples are allocated differently
Are nuisance effects confounded with treatment effects now?
Auer & Doerge, Genetics 185:405, 2010

7. Experimental design No –
biological replicates are randomized across flowcells, and treatments are randomized across lanes. Biological sources of variation (reps) are orthogonal to technical sources (flowcells and lanes).
Auer & Doerge, Genetics 185:405, 2010

8. Map reads to reference genome or transcriptome
Design experiment
Prepare libraries
Conduct sequencing
Produce FASTQ files
Pre- process data
Auer & Doerge, 2010
Genetics 185:405-16
DNA sequences A
DNA sequences B
Fu et al, 2014,
PNAS 111:1891-6
Map reads to reference genome or transcriptome

10. Molecular indexing measures bias
Abundant transcripts are detected more times than there are unique start/stop sites for sequence reads
Fu et al, PNAS 111:1891–1896, 2014

11. The more steps, the greater the sample loss…
Even with estimated 70% yield at each step, the cumulative yield after 15 steps in RNA-seq library production is very low
Fu et al, PNAS 111:1891–1896, 2014

12. Map reads to reference genome or transcriptome
Design experiment
Prepare libraries
Conduct sequencing
Produce FASTQ files
Pre- process data
Auer & Doerge, 2010
Genetics 185:405-16
DNA sequences A
DNA sequences B
Fu et al, 2014,
PNAS 111:1891-6
Map reads to reference genome or transcriptome

13. Retrieve genome annotation from database A A B B
DNA sequences A
DNA sequences B
Map reads to reference genome or transcriptome
Merge read alignment with genome annotation to produce tables with counts of aligned reads per genome feature
BAM file A
Count table A
BAM file B
Count table B

14. Table of differentially-expressed genes with annotation
Genome annotation from database
Merge read alignment with genome annotation to produce tables with counts of aligned reads per genome feature
Count table A
Statistical comparison of counts, inference of differential expression
Count table B

15. Align reads to reference
Assemble transcripts
Merge across treatments
Count reads per treatment for assembled transcripts, adjusting for bias detected in start- or end-points of RNA fragments to more accurately estimate transcript levels (Roberts et al., Genome Biol 12:R22, 2011)
Count reads per treatment for assembled transcripts
Visualize differential expression results
From