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Jason Williams Cold Spring Harbor Laboratory, DNA Learning Center [email protected] @JasonWilliamsNY DNALC Live Intro to RNA-Seq with Jupyter Part II

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Page 1: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Jason WilliamsCold Spring Harbor Laboratory, DNA Learning Center

[email protected]@JasonWilliamsNY

DNALC Live

Intro to RNA-Seq with JupyterPart II

Page 2: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

DNALC LiveThis is an experiment, give us feedback

on what you would like to see!

Page 3: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

dnalc.cshl.edu

DNALC Website and Social Media

dnalc.cshl.edu/dnalc-live

Page 4: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

youtube.com/DNALearningCenter

DNALC Website and Social Media

facebook.com/cshldnalc

@dnalc

@dna_learning_center

Page 5: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Who is this course for?

• Audience(s): • Undergraduate biology 200 level and up • (advanced AP Bio/graduate)

• Format: 2 sessions (1 per week); ~ 45 minutes each

• Exercises: Follow along through CyVerse

• Learning resources: Slides and online lesson available

Page 6: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Course Learning Goals• Understand the rationale of an RNA-Seq experiment and its design

• Learn about the Linux command line

• Use Jupyter (SRA Toolkit) to import sequence data

• Use Jupyter (FastQC/Trimmomatic) to quality check/trim sequence data

• Use Jupyter (Kallisto) to (pseudo)align reads

• Use Jupyter (genomeview/UCSC) to explore RNA-Seq results

Page 7: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Lab Setup• We will be using CyVerse VICE – You can get a free account at cyverse.org

(required)

Page 8: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Intro to RNA-Seq with JupyterPart II

(alignment and visualization)

Page 9: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Steps for today’s session

• Review data set and data cleaning

• Learn about alignment of reads

• See how to visualize data in a genome browser

Page 10: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

RNA-Seq with DNA Subwaydnalc.cshl.edu/dnalc-live

Page 11: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Introduction to RNA-Seq

Page 12: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

What is RNA-Seq? - measuring the transcriptome

• To understand what genes are active, and under what circumstances, we must know what genes are being transcribed into messenger RNA

Page 13: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

What is RNA-Seq? - measuring the transcriptome

• To understand what genes are active, and under what circumstances, we must know what genes are being transcribed into messenger RNA

• A cell in the liver has the same DNA instructions as a neuron in the brain. However the genes being expressed differ greatly between these cells

Page 14: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

What is RNA-Seq? - measuring the transcriptome

• RNA-Seq allows us to measure the transcriptome – take an account of all transcription occurring in a cell/tissue

Page 15: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

What is RNA-Seq? - measuring the transcriptome

• RNA-Seq allows us to measure the transcriptome – take an account of all transcription occurring in a cell/tissue

• We use the abundance of an RNA transcript as a proxy for the activity of some cellular process (e.g. protein synthesis, regulatory activity)

Page 16: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

What is RNA-Seq? - measuring the transcriptome

• RNA-Seq allows us to measure the transcriptome – take an account of all transcription occurring in a cell/tissue

• We use the abundance of an RNA transcript as a proxy for the activity of some cellular process (e.g. protein synthesis, regulatory activity)

• We analyze these data to compare samples (e.g. cancerous vs. non-cancerous)

Page 17: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

What is RNA-Seq?

Page 18: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Introduction to our data set

Page 19: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

An NSF Research Collaboration Network

Page 20: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Leptin expression vs. diet – RNA-Seq pilot lesson

Colon tissue/tumors in mice raise on Regular (RD) or High-fat (HFD) diet

Page 21: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Leptin expression vs. diet – RNA-Seq pilot lesson

Colon tissue/tumors in mice raise on Regular (RD) or High-fat (HFD) diet

Page 22: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Sequence data from NCBI

https://www.ncbi.nlm.nih.gov/bioproject/PRJNA353374

https://www.ncbi.nlm.nih.gov/bioproject/PRJNA353374
Page 23: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Access lessons and sign in on CyVerse

Page 24: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Phred scores…

Phred Score Error (bases miscalled) Accuracy10 1 in 10 90%20 1 in 100 99%30 1 in 1,000 99.9%40 1 in 10,000 99.99%50 1 in 100,000 99.999%

Page 25: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Lab – Sequence alignment

Page 26: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

There are many roads to RNA-Seq

Photo credit:Conesa et al. Genome Biology (2016) 17:13DOI 10.1186/s13059-016-0881-8

Page 27: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

RNA-Seq with Kallisto

Page 28: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

RNA-Seq with Kallisto

Kallisto (pseudo)aligns reads to a reference transcriptome

1. An index is built of the reference transcriptome

2. Sequence reads are (pseudo)aligned to transcripts

Page 29: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Reference transcriptome

A collection of “all” the transcripts in an organism

Ensembl tour: https://useast.ensembl.org/Homo_sapiens/Info/Index

Page 30: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Kallisto – Pseudoalignment

Photo credit:http://mcb112.org/w02/w02-lecture.html

Page 31: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Kallisto results

Page 32: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Kallisto results

• target_id: Identifier for the transcript (from Ensembl)

Page 33: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Kallisto results

Page 34: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Kallisto results

• target_id: Identifier for the transcript (from Ensembl)

• length: length (nucleotides) of transcript exons

Page 35: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Kallisto results

• target_id: Identifier for the transcript (from Ensembl)

• length: length (nucleotides) of transcript exons

• eff_length: length of transcript that was sampled*

*In the original sequencing library, we rarely sample whole entire transcripts, this number accounts for the fragment length of the library

Page 36: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Kallisto results

• target_id: Identifier for the transcript (from Ensembl)

• length: length (nucleotides) of transcript exons

• eff_length: length of transcript that was sampled*

• est_counts: The estimated number of reads that have mapped to the transcript

*In the original sequencing library, we rarely sample whole entire transcripts, this number accounts for the fragment length of the library

Page 37: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Normalization – gene length

Gene A Gene B

Which is longer (bp)?

Page 38: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Normalization – gene length

Gene A(300bp)

Gene B (100bp)

Which has more reads?

Page 39: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Normalization – gene lengthWhich has more reads?

Gene A(300bp)

Gene B (100bp)

Page 40: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Normalization – gene length

1627Gene A(300bp)

Gene B (100bp)

Page 41: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Normalization – gene length

Gene A(300bp)

27/300 = 0.09

Gene B (100bp)

16/100 = 0.16

1627

Page 42: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

Goal recap• Understand the rationale of an RNA-Seq experiment and its design

• Learn about the Linux command line

• Use Jupyter (SRA Toolkit) to import sequence data

• Use Jupyter (FastQC/Trimmomatic) to quality check/trim sequence data

• Use Jupyter (Kallisto) to (pseudo)align reads

• Use Jupyter (genomeview/UCSC) to explore RNA-Seq results

Page 43: Intro to RNA-Seq with Jupyter Part II · • Understand the rationale of an RNA-Seq experiment and its design • Learn about the Linux command line • Use Jupyter (SRA Toolkit)

dnalc.cshl.edu

DNALC Website and Social Media

dnalc.cshl.edu/dnalc-live


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