RNA Sequencing: Workflow Recommendations
RNA Sequencing: Workflow RecommendationsSample Preparation – back to the basicsSridar Chitturschittur@albany.edu(518) 591-7215
RNA isolation processesCollection& StabilizationTissue Disruption& HomogenizationRNA Problems are not usually at the extraction step butbefore and after Treatment and Handling- Collection method?- Disruption technique? Storage– How will you store your isolated RNA2
Characteristics of RNA Hydrophilic: It dissolves in water. It does not dissolve in phenolor alcohol or lipids pH: RNA is unstable in a basic environment (pH 9 or more). It isstable in mild acid (pH 4) but not strong acid (pH 1). Therefore,try to keep at neutral pH Heat: Fairly stable up to 65 C if divalent cations are not present Absorbance Spectra : Maximum UV absorbance at 260nm3
RNA DegradationMg HeatThe OH- group attacks the nearby phosphate, breaking the phosphate backboneThis is catalyzed by high pH, divalent cations plus heat, RNases, etc.4
RNA : sensitive nucleic acidRNases Found just about everywhere: the benchtop, your hands, in the air and more. Incredibly stable enzymes (can withstand extreme conditions such as heatand high salt) it readily renatures and retains activity Some are sequence-specific, others are not. RNase A, for example, cuts 3’end of unpaired C and U residues Are not destroyed by autoclaving. They must be destroyed with DEPC orRNaseZAP or inactivated with RNase inhibitors like SUPERase-In 5RNase A molecule
10 ways to improve RNA Isolation6
General rules on RNA yieldsRNAses quantity in the mouse organs181 000x7
1. Reduce exposure to environmental RNases Essential that any items that could contact the purified RNA isRNase-free Benchtop, Pipettors, Glassware should be decontaminatedwith RNAseZap RNase free tips, tubes and solution should always be used andgloves should be changed frequently8
2. Immediately inactivate endogenous,intracellular RNases.There are 3 effective methods to accomplish this: I.II.III.Homogenize samples immmediately after harvesting in achaotropic-based cell lysis solution (e.g. Guanidinium)Flash freeze in liquid nitrogen. Tissue pieces must be smallPlace sample in RNAlater 9
3. Use proper cell or Tissue storage conditions If flash frozen, must be stored at -80 C and never allowed tothaw prior to homogenization and isolation.10
4. Thoroughly homogenize samples Essential step to prevent both loss and degradationNeeds to be fast and thoroughMethod should be tailored to the cell typeUsually Mechanical:– Dounce, polytron, vortex, sonication, French press, coffee grinder, bar shaker. or Enzymatic– Lysozyme, zymolase and lysostaphin etc.11
5. Pretreat homogenate before RNA Isolation Needed for some samples after homogenization before isolationto increase RNA yield– High fat contain tissues, like Brain or Adipose tissue should beextracted with Chloroform to remove lipids– Plant tissues high in polyphenolics and polysaccharides should bepretreated with insoluble polyvinylpyrrolidone (PVP)12
6. Choose the best RNA isolation method Sample type– Tissues, blood, cells, FFPE, LCM, Bacteria or yeast What is the downstream application– NGS, Microarray, qRT-PCR, Northern How many samples ? Other Isolation Needs– Viral RNA, microRNAs, mRNA isolation/enrichment13
Methods in RNA IsolationTotal RNA IsolationSolid Phase/GlassFilter MethodologyMagnetic BeadMethodsCombination ofAll the above14Organic ExtractionPhenol-basedMethod
7. DNase treatment Recommended when RNA is to be used in Transcriptome orGene ST arrays Recommended for WT RNAseq when ribodepletion is used Good practice when isolating RNA from DNA rich tissues likespleen15
8. How to precipitate? If purified RNA needs to be concentrated byprecipitation for downstream applications:– Ammonium Acetate (NH4OAc) precipitation– For low concentrations of RNA (ng/ml) use glycogen, pelletpaint16
9. Resuspension Three ideal qualities of a resuspension solution–RNase-free water (for NGS studies)–Low pH (pH 6-7)–Incorporate a chelating agent to protect RNA againstdegradation by introduced RNases sodium citrate 1 mM, pH 6.4 0.2 or EDTA 0.1mM or TE buffer 10mM Tris-HCl, 1 mM EDTA, pH 7.017
10. Storage Short term:– Stored at -20 C Long term:– Stored at -80 C in buffer– More stable in NH4OAc/EtOH precipitation mixture at -80 C Aliquot your RNA into several tubes– Avoid Freeze-thaw event– Prevent RNase contamination18
Sample QC19
NanodropRNA QC is critical to the success of your genomics experiment
The Nanodrop can reveal problematic samplesSample is NOT 184 ng/ulActually 48 ng/ul as per Qubit/Bioanalyzer272 nm peak is skewing data260RNATRZΔ 1.2OD 48 ng/ul272How will thisaffectdownstreamprocesses suchas RT-qPCR ifone assumesequal RNA inputto a cDNAreaction?
The Nanodrop does not tell the whole storyGood RNADegraded RNAGenomic DNA
Nucleic acid quantitation using fluorescenceNote: these do not give any information on RNA integrity or protein contamination
Measuring RNA integrityRNA QC is critical to the success of your genomics experiment
Capillary electrophoresisAgilent BioAnalyzer 2100Biorad Experion
The Agilent Expert software assigns a RIN number to each trace. It assigns a numberaccording to how much signal is found between the 5S and 18S band, between the 18S and28S bands, and after the 28S band. A RIN number of 10 is perfect score. The software doesnot always call RIN numbers for prokaryotic RNA and the RIN can be misleading forsamples containing additional RNA bands such as those from chloroplasts or a symbioticRNA.
28sSmall RNA RegionLower Marker18sSmall RNA chip versus existing RNA chipRIN: 8.1RNA 6000NanoSize range: 25-6000ntResults: Integrity, Total RNA amount, gDNAcontaminationRNA 6000Nano kitSmall RNA Kit5.8s5smiRNA RegionSmall RNASize range: 6-150nttRNALower MarkerSmall RNA RegionResults: miRNA amount, Ratio and amount ofother Small RNA28
High Sensitivity DNA Assay
DNA high sensitivity assay: Bioanalyzer QC (qualitative)
Realistic quantitative estimation
SPRI bead selection Each bead is made of polystyrene surrounded by a layer of magnetite, which iscoated with carboxyl molecules. Beads reversibly bind DNA in the presence of the “crowding agent” polyethyleneglycol (PEG) and salt (20% PEG, 2.5M NaCl is the magic mix). PEG causes thenegatively-charged DNA to bind with the carboxyl groups on the bead surface. As the immobilization is dependent on the concentration of PEG and salt in thereaction, the volumetric ratio of beads to DNA is critical The lower the ratio of SPRI:DNA the higher larger the final fragments will be atelution
Library prep kit choices
Illumina Truseq stranded library prepmRNA kit: 0.1-4ug oftotal RNA or 10400ng of mRNATotal kit: 0.5 – 1ug total RNA
Illumina Truseq stranded library prep
Illumina Truseq stranded library prep
Not enough total RNA?Other choices:Clontech smarter kit NexteraClontech low input kitBioo ScientificNEB
SMARTer Stranded Total RNA-Seq Kit- Pico Input Mammalian (Clontech)Recommended input ranging from 250 pg to 10 ng of totalmammalian RNAIncorporates SMART (Switching Mechanism At 5' end of RNATemplate) technology and locked nucleic acid (LNA) technology.PCR amplification generates Illumina-compatible libraries withoutthe need for adapter ligation.The directionality of the template-switching reaction preservesthe strand orientation of the original RNA, making it possible toobtain strand-specific sequencing data from the synthesizedcDNA.The ribosomal cDNA (originating from rRNA) is then cleaved byZapR in the presence of the mammalian-specific R-Probes
SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian(cont’d)Successful cDNA synthesis and amplification shouldproduce a distinct curve spanning 200–1,000 bp, with alocal maximum at 300–400 bpRead 1 is derived from the sense strand of the input RNAFirst three bases are from TSO and must be trimmedRead 2 will correspond to the antisense strand (PE reads)
SMARTer Ultra Low Input RNA Kit Nextera XT(Clontech Illumina)high-quality cDNA directly from 1–1,000 cells or 10 pg–10 ng of total RNAUse the Nextera XT DNA Sample Preparation Kitfrom Illumina to prepare your library. We recommendusing an input amount of 100–150 pg amplified DNA.Sequence only from Read Primer 1. (i.e.1x75bp)
NEBNext Ultra Directional RNA Library Prep Kit(New England Biolabs)Total RNA (100 ng–1 μg)If a peak at 80 bp (primers) or 128 bp (adaptor-dimer)
NEXTflex Rapid Directional RNA-Seq Kit (Bioo Scientific)10 ng - 1 μg of total RNA 2 hours faster than traditional Illumina stranded RNA-Seq libraryprep protocols Provides precise measurement of strand orientation ( 99%) 10 ng – 1 µg total RNA for enrichment by NEXTflex Poly(A)Beads or 1 ng - 100 ng isolated mRNA or rRNA-depleted RNA Minimal hands-on time required Streamlined protocol reduces sample loss Utilizes dUTP-based methodology Robust, reliable performance Up to 96 barcodes available for multiplexing Automation protocols are available for the Sciclone NGS andNGSx Workstation Functionally validated with Illumina sequencing platforms Allows for single read or paired end transcriptome sequencing
RNA library prep methods:Illumina: Truseq small RNA1ug total RNA, 10-50ng purified miRNA
CFG process flow
PI contactsCFG for NGSbyemail/phoneContact PI,provideupdatesInitialconsultation todetermine scopeand deadline ofworkLibrary prepand QC 2Contact PI,provide updatesSequencelibrariesand QC 3Identifyissues andtroubleshootCFG staffidentifiesprotocolandresearcheskitsContact PI,beginprotocolsPricingdeterminedand quotesentSamplesundergo qualitycheck 1Quoteacceptedand CFGprocuresmaterialsSamplesandmaterialsreceived byCFGContact PIObtain newsamples orredefine projectIdentify issuesandtroubleshootContact PI with data1: RNA Bioanalyzer, nanodrop2: HS DNA chip bioanalyzer,nanodrop, qubit3: Run metrics check, FastQC
RNA seq3’ end counting(poly A, gene expression)TruseqClontech/NexteraotherWhole transcriptome(gene expression,splicing, lncRNA)Clontech picoTruseqNEB0.1-4ug totalRNA or 10400ng mRNA10pg-10ng total RNA0.1-1ug total RNA0.1-1ug total RNA10ng-100ngreduced RNASequencing TypeSequencing parametersDifferential gene expression1x75 20-25M reads1x150 20-25M readsAlternative splicing, WT, lncRNA2x75 50-60M reads2x150 50-60M readsSmall RNA1x75 2-5M readsscRNA seqPE 26, 110 5-10M per cell250pg-10ng total RNAClontech HI100ng-1ug total RNA
Illumina Current specificationsMiseqNextseq 500Hiseq 2500Hiseq 3000Hiseq 4000Output Range0.5 - 15 Gb20 - 120 Gb10 - 1000 Gb125 - 750 Gb125 - 1500 GbMaximum Read Length2 x 300 bp2 x 150 bp2 x 150 bp2 x 150 bp2 x 150 bpReads per Run15 million130 - 400 million300 million - 4 billion2.5 billion2.5 - 5 billionRun Time4hr - 55 hr11 hr - 29 hr7 hr - 6 days 1 - 3.5 days 1 - 3.5 daysKey MethodsSmall genome, amplicon, &targeted gene panelsequencing.Exome, transcriptome, &whole-genome sequencing.Exome, transcriptome, &whole-genome sequencing.Exome, transcriptome, &whole-genome sequencing.Exome, transcriptome, &whole-genome sequencing.Hiseq X: 1.8Tb output, 6 B reads in 3 days
NextSeq Performance Parameters Total times include cluster generation, sequencing and base calling on aNextSeq 500 system Install specifications are based on Illumina PhiX control library atsupported cluster densities (between 170 and 230 k/mm2 clusterspassing filter) The percentage of bases Q30 (1/1000 chance of an incorrect baseidentification or 99.9% accuracy) is averaged over the entire run
Run Quality checks
Illumina Basespace Dashboard/ SAVIntensity (P90): The 90% percentile extracted intensity for a given image (lane/tile/cycle/channel combination).FWHM The average full width of clusters at half maximum (representing their approximatesize in pixels).
Q 10 log10 P
CV 25% between samplesAllows to see how the reads are spread between the pooled samples
Surface view of the flow cellBlue regions low sequencing quality
Other ways to look at Q scores
Error rate goes up with cyclesbut is still very low% Base: The percentage ofcalled (non-N) clusters forwhich the selected base hasbeen called.
Run metricsCycles 25 through run completion: Intensity, Cluster Density, % PF, Yield, Q scoresExpected: 170-230 K/mm2Q30 80% 1x75, 2X75; Q30 75% 2x150RPF 400M SE, 800M PE (HO, 130M SE, 260M PE (MO)
Data Quality Checks
Q 10 log10 PQ30: 1 in 1000 errorFor each position a BoxWhisker type plot is drawn. The elements of the plot are as follows: The central red line is the median value The yellow box represents the inter-quartile range (25-75%) The upper and lower whiskers represent the 10% and 90% points The blue line represents the mean qualityA warning will be issued if the lower quartile for any base is less than 10, or if themedian for any base is less than 25.This module will raise a failure if the lower quartile for any base is less than 5 or if themedian for any base is less than 20.
The per sequence quality score report allows you to see if a subset of your sequences have universallylow quality values. It is often the case that a subset of sequences will have universally poor quality, oftenbecause they are poorly imaged (on the edge of the field of view etc), however these should representonly a small percentage of the total sequences.A warning is raised if the most frequently observed mean quality is below 27 - this equatesto a 0.2% error rate.An error is raised if the most frequently observed mean quality is below 20 - this equates toa 1% error rate.
This module issues a warning if the difference between A and T, or G and C is greater than10% in any position.This module will fail if the difference between A and T, or G and C is greater than 20% in anyposition.
This module issues a warning it the GC content of any base strays more than 5% from themean GC content.This module will fail if the GC content of any base strays more than 10% from the mean GCcontent.
A warning is raised if the sum of the deviations from the normal distribution representsmore than 15% of the reads.This module will indicate a failure if the sum of the deviations from the normal distributionrepresents more than 30% of the reads.
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Truseq. Clontech/Nextera. other. Truseq. NEB. Clontech pico. 0.1-4ug total RNA or 10-400ng mRNA. 10pg-10ng total RNA. 0.1-1ug total RNA. 0.1-1ug total RNA. 10ng-100ng reduced
The SMARTer Universal Low Input RNA Kit allows high-quality cDNA synthesis starting from as little as 200 pg of input RNA. The kit has been validated to prepare cDNA samples for sequencing and RNA expression analysis with next-generation sequencing instruments. The entire library construction protocol can be completed in two days (Figure 1).
(Structure of RNA from Life Sciences for all, Grade 12, Figure 4.14, Page 193) Types of RNA RNA is manufactured by DNA. There are three types of RNA. The three types of RNA: 1. Messenger RNA (mRNA). It carries information about the amino acid sequence of a particular protein from the DNA in the nucleus to th
The process of protein synthesis can be divided into 2 stages: transcription and translation. 5 as a template to make 3 types of RNA: a) messengermessenger--RNA (mRNA)RNA (mRNA) b) ribosomalribosomal--RNA (rRNA)RNA (rRNA) c) transfertransfer--RNA (tRNA)RNA (tRNA) Objective 32 2)2) During During translationtranslation, the
10 - RNA Modifications After the RNA molecule is produced by transcription (Part 9), the structure of the RNA is often modified prior to being translated into a protein. These modifications to the RNA molecule are called RNA modifications or posttranscriptional modifications. Most RNA modifications apply onl
13.1 RNA RNA Synthesis In transcription, RNA polymerase separates the two DNA strands. RNA then uses one strand as a template to make a complementary strand of RNA. RNA contains the nucleotide uracil instead of the nucleotide thymine. Follow the direction
DNA AND RNA Table 4.1: Some important types of RNA. Name Abbreviation Function Messenger RNA mRNA Carries the message from the DNA to the protein factory Ribosomal RNA rRNA Comprises part of the protein factory Transfer RNA tRNA Transfers the correct building block to the nascent protein Interference RNA
Next-Gen Sequencing Workflow Source: Lu and Shen, 2016, Biochemistry, Genetics and Molecular Biology. DOI: 10.5772/61657 Genome Whole genome sequencing Whole exome sequencing Targeted gene panels (cancer, newborns, autism, etc.) Transcriptome Whole RNA sequencing mRNA transcriptome (poly-A selection)
Abrasive Jet Micro Machining (AJMM) is a relatively new approach to the fabrication of micro structures. AJMM is a promising technique to three-dimensional machining of glass and silicon in order to realize economically viable micro-electro-mechanical systems (MEMS) It employs a mixture of a fluid (air or gas) with abrasive particles. In contrast to direct blasting, the surface is exposed .
