An introduction to RNA-Seq. Brian J. Knaus USDA Forest Service Pacific Northwest Research Station
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1 An introduction to RNA-Seq Brian J. Knaus USDA Forest Service Pacific Northwest Research Station 1
2 Doerge, R.W Nature Reviews Genetics 3:
3 An introduction to RNA-Seq Tissue collection RNA extraction Library preparation Illumina sequencing Read mapping Statistical hypothesis testing 3
4 Tissue collection Circadian rhythm! RNA isolation 4
5 Coos Bay P.m. var. menziesii Yakima P.m. var. glauca Sample#1 6,036,336 reads Sample#2 8,029,643 reads Sample#1 4,900,955 reads Sample#2 4,580,341 reads Experimental design * Each sample is a pool of six seedlings grown in a common environment. 5
6 RNA-Seq library prep mrna AAAAAA TTTTTT Paramagnetic bead Purify RNA X2 mrna AAA AAAAAA Fragment RNA (Mg + heat; ~200 bp) mrna AAA AAAAAA DNA 5 DNA-5 DNA-5 First strand synthesis Random hexamers 5 -DNA 5 -DNA 5 -DNA DNA 5 DNA-5 DNA-5 Second strand synthesis 6
7 Library prep 5 -DNA 5 -DNA 5 -DNA DNA 5 DNA-5 DNA-5 End repair A 5 -DNA A 5 -DNA A 5 -DNA A DNA 5 A DNA-5 A DNA-5 A overhang Adapter T A 5 -DNA DNA-5 A T Adapter Ligate adapters Size select electrophoresis PCR Enrichment 7
8 Metzker, M.L Nature Reviews Genetics 11:
9 9
10 One Illumina tile. Four pictures (one per nucleotide) per cycle. Each lit pixel is a cluster or micro-read. 10
11 Seven lanes (100 tiles each) of a flowcell Fc154 11
12 Fastq CGTTNCCACTTCCTCCATCATGTCATCATGTGCGACAGGA +HWI-EAS121:1:1:0:952#0/1 CGTTNTAAAGGTGCACCAGGGATCAAATCAATGGAATGCT +HWI-EAS121:1:1:0:405#0/1 aa^[dva^`^_y`a^a`[\^\z^aayz`a`x CGTTNCATGCCCTTCTTTAATTTTTACACATGGTTCTTCT +HWI-EAS121:1:1:0:724#0/1 TTGTNAAAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG +HWI-EAS121:1:1:0:666#0/1 a`bod[]r]`a TTGTNCTCACCTATAATTTGACTTTGACATGCTACCTAGC +HWI-EAS121:1:1:0:1591#0/1 aaayd[aaa`aaaawzaaaaa``_aaaa`aa`_v_``y[a 40-mer sequences 12
13 Read mapping Requirements: Reference Micro-reads Software: Bowtie (Tophat) BWA CASHX Eland (Illumina) CLC Bio 13
14 MAQ reference based assembly 14
15 MAQ reference based assembly 15
16 Gene cb_a cb_b yk_a yk_b isotig18613_gene=isogroup07808_length=677_numcontigs= isotig01880_gene=isogroup00225_length=652_numcontigs= isotig07160_gene=isogroup01638_length=3698_numcontigs= isotig06362_gene=isogroup01321_length=1396_numcontigs= isotig06005_gene=isogroup01197_length=1204_numcontigs= isotig06363_gene=isogroup01321_length=1470_numcontigs= contig29123_gene=isogroup00629_length= isotig30058_gene=isogroup19254_length=1101_numcontigs= contig50604_gene=isogroup01657_length= contig21101_gene=isogroup01657_length= isotig05419_gene=isogroup01011_length=1938_numcontigs= contig03433_gene=isogroup00629_length= isotig05877_gene=isogroup01156_length=2570_numcontigs= isotig04630_gene=isogroup00778_length=2155_numcontigs= isotig29494_gene=isogroup18690_length=2738_numcontigs= contig46195_gene=isogroup00001_length= contig48481_gene=isogroup00006_length= isotig30667_gene=isogroup19863_length=1653_numcontigs= isotig33299_gene=isogroup22495_length=1270_numcontigs= isotig24557_gene=isogroup13753_length=1317_numcontigs=
17 Hypothesis Testing Requirements: Table of read counts Model: Y ~ Negbinom(μ,φ) Issues: Normalization for unequal library size Estimate of dispersion parameter (φ) Estimate of rate parameter (μ) Test for differential expression (exact test) Correct for multiple comparisons 17
18 Hypothesis Testing Software: EdgerR (Bioconductor) Genecounter (in prep) Cufflinks CLC Bio 18
19 Coos Bay P.m. var. menziesii Yakima P.m. var. glauca Sample#1 6,036,336 reads Sample#2 8,029,643 reads Sample#1 4,900,955 reads Sample#2 4,580,341 reads Experimental design * Each sample is a pool of six seedlings grown in a common environment. 19
20 Yakima, WA Coos Bay, OR 20
21 Gene cb_a cb_b yk_a yk_b isotig18613_gene=isogroup07808_length=677_numcontigs= isotig01880_gene=isogroup00225_length=652_numcontigs= isotig07160_gene=isogroup01638_length=3698_numcontigs= isotig06362_gene=isogroup01321_length=1396_numcontigs= isotig06005_gene=isogroup01197_length=1204_numcontigs= isotig06363_gene=isogroup01321_length=1470_numcontigs= contig29123_gene=isogroup00629_length= isotig30058_gene=isogroup19254_length=1101_numcontigs= contig50604_gene=isogroup01657_length= contig21101_gene=isogroup01657_length= isotig05419_gene=isogroup01011_length=1938_numcontigs= contig03433_gene=isogroup00629_length= isotig05877_gene=isogroup01156_length=2570_numcontigs= isotig04630_gene=isogroup00778_length=2155_numcontigs= isotig29494_gene=isogroup18690_length=2738_numcontigs= contig46195_gene=isogroup00001_length= contig48481_gene=isogroup00006_length= isotig30667_gene=isogroup19863_length=1653_numcontigs= isotig33299_gene=isogroup22495_length=1270_numcontigs= isotig24557_gene=isogroup13753_length=1317_numcontigs=
22 Hypothesis Testing Requirements: Table of read counts Model: Y ~ Negbinom(μ,φ) Issues: Normalization for unequal library size Estimate of dispersion parameter (φ) Estimate of rate parameter (μ) Test for differential expression (exact test) Correct for multiple comparisons 22
23 23
24 Y ~ Pois(λ) 24
25 25
26 Issues? 26
27 Product #2 Differential Expression (RNA-Seq) 27
28 28
29 Random hexamer bias Random hexamer #2 Oligo dt #2 29
30 30
31
32
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