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1 Center for Reproductive Genomics Department of Biomedical Sciences College of Veterinary Medicine Cornell University Jen Grenier
2 Compute power Software/parameters Reference genome Data management Data Analysis Testable Hypothesis Experimental Design Plan ahead Instrument Read length Single/Paired NGS Sequencing Sample Preparation Replicates Input amount Quality NGS Library Preparation Library type Barcoding Research:Technology Partnership
3 Transcriptome sequencing (RNAseq) total RNA polya + mrna total RNA rrna-subtracted RNA viral RNA custom input Small RNA sequencing total RNA microrna, sirna, pirna All-inclusive Packages One price includes library prep, sequencing, and analysis Single point of contact Project management focus
4 All-inclusive Project Services Single Point of Contact Experimental design RNA sample preparation/qc Library preparation - barcoding - QC - pooling Illumina sequencing Standard analysis - data QC, preprocessing - genome/transcriptome mapping - normalization - differential expression Custom analysis Validation, follow-up Cornell Pricing CVM labs NextSeq (75nt reads) 20M reads/library $370 $265 Add l 20M reads $115 $115 rrna removal: Ribo-Zero HMR $100 $100 10M reads/library $340 $232 Add l 10M reads $80 $80
5 yes RNA Quality RQN 7 no yes polya + OK no yes 1 µg total RNA no yes 100 ng total RNA no yes eukaryote no discuss Directional (stranded) RNAseq Standard (nonstranded) RNAseq 100 ng ng PolyA + RNAseq RQN 7 and polya + OK Illumina Instrument: HMR rrna - (stranded) RNAseq eukaryote Other rrna - (stranded) RNAseq bacteria/mix rrna - RNAseq RQN < 7 or polya + not OK NextSeq500 lowest cost and fastest turn-around time
6 Experimental Design minimize batch effects, other variables test protocols, then be consistent Quality Control sample quality library quality data quality biological signal purity, integrity yield, insert size range read counts/quality, mapping rates, biases sample clustering, DE genes
7 Isolation Method Trizol - highest yield, requires phase extraction/precipitation Silica spin column - watch out for small RNA recovery Concentration/Yield Nanodrop (A260) - less sensitive, but also gives curve/purity ratios Qubit (fluorescence) - more sensitive, use for samples <20ng/ul Chemical Purity Nanodrop shape of curve (ratios) indicate salts/organics/protein Biological Integrity Bioanalyzer QC - RIN/RQN
8 Sample Collection Lysis Conditions sample handling storage cryofreeze (dry) homogenize in lysis buffer/freeze or prep RNAlater (fridge O/N) rapid, at cellular level fresh: homogenize in lysis buffer cryofrozen: grind on dry ice or homogenize in lysis buffer keep RNA samples on ice at all times don t vortex purified RNA (avoid shearing) store at -80, minimize freeze-thaw cycles
9 Pure Nucleic Acid High A270 High A230, shifted trough Concentration: α A260 (RNA extinction coeff = 40) Purity: A260/A low: chaotropic salt contamination A260/A low: protein (A280) or phenol (A270) contamination
10 RQN 7 : polya + RNAseq RQN < 7 : riborna - RNAseq RQN 9.7 RQN 4.9 RQN 6.8 RQN 1.2
11 Genomic DNA can be co-purified with RNA identified by RNA QC (high MW) or RNA-specific quantification (Qubit) vs nanodrop resolved by (RNAse-free) DNAse Tx
12 Yield Chemical purity improve lysis efficiency try Trizol instead of column clean up existing RNA sample repeat RNA isolation Biological integrity change sample collection procedure improve lysis efficiency/speed subtract rrna instead of enriching mrna
13 Raw data QC FastQC: base quality scores, overrepresented sequences Standard analysis Preprocessing Trim adaptor sequences Filter low-quality, short, and contamination sequences Genome mapping Gene expression quantification Annotated genes (or micrornas) Additional analyses Hierarchical clustering Principal component analysis Gene set analysis GO enrichment, pathways, GSEA novel transcript and mirna prediction mrna microrna interactions
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