BioMicroCenter:RNA LIB

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Wang ET, et al. Nature 2008

The BioMicro Center supports a broad variety of standard library preparation methods for RNAseq. The choice of method is highly dependent on the type of input, the amount of input RNA available, and the quality of the input RNA. The key in all RNAseq methods is the avoidance of ribosomal RNA, which would typically dominate the library preparation. Below area summary of the methods we utilize routinely in the core. For High-Throughput RNA library preparation, please check out our new page for methods designed specifically for large sample batches.

Please note some methods are currently in transition to try to improve data quality and reduce library preparation costs.

Amount of RNA	Quality of RNA	Method Recommended
>25ng	RIN:9.0	Kapa Hyperprep Illumina NeoPrep or Illumina Truseq (>100ng) - Retired 7/17
>1ug	RIN:5.0	Kapa RiboErase Epicenter RiboZero - only for non-human/mouse
10pg-25ng	RIN:9.0	Clontech SMARTer v4
1ng-1ug	RIN:5.0	Clontech Pico Ribosomal Depletion (ZapR).
smallRNA	NA	BIOO SmallRNA Kit or Qiagen miRNA kit.

Kapa mRNA Hyperprep

With the retirement of the Neoprep, the BioMicro Center is converting standard library preparation over to the Kapa mRNA hyperprep kit. This workflow is very similar to Illumina's TruSeq chemistry at lower cost and is streamlined for automation. This chemistry uses polyT beads to isolate the mRNA from the rRNA and tRNA. The use of these beads requires that the RNA be of very high quality or only the 3' end of transcripts will be isolated. Purified mRNA is then fragmented with metal and random priming is used to convert the sample to cDNA. Once double-stranded cDNA is generated, LMPCR is performed to create the indexed Illumina library. The BioMicro Center will be offering mRNA HyperPrep as a single sample reaction or in batches of 24 done on the TecanEvo 150s. We are also exploring reduced volume reactions using the Mosquito HV to further reduce costs.

TruSeq Chemistry

Sample Data

Kapa RiboErase & EpiCenter RiboZero

For samples with degraded RNA or samples where you are interested in looking at non-polyA RNAs, the BioMicro Center utilizes the Kapa RNA RiboErase for Human/Mouse samples and Epicenter RiboZero kit for other species. RiboErase uses RNAseH to degrade rRNAs while RiboZero uses magnetic beads coupled to rRNA sequences to remove these sequences from the solution. The remaining mRNA fragments can then be converted in to cDNA and prepared using the Kapa mRNA Hyperprep kit to produce the Illumina library.

Clontech SMARTer Low-Input

For samples with less then 100ng of input, the BioMicro Center utilizes the Clontech SMARTer system. This system differs from the TruSeq chemisry in that it begins with cDNA generation using polyT priming and proprietary chemistry. The use of polyT priming requires the RNA to be of high quality. Full length double-stranded cDNAs are generated and amplified by PCR. These cDNAs are then fragmented and transferred to the SPRIworks for the remainder of sample preparation. Data from this system is of similar quality to samples created with Illumina TruSeq chemistry.

Clontech system. Image from Clonetech

NuGEN Ovation RNAseq System V2

For samples with less then 100ng of input and restricted input amounts, our kit of choice is the NuGEN Ovation. This kit utilizes non-random nonamers designed to not amplify ribosomal RNA to create double stranded cDNA fragments. The fragmented cDNA is then transferred to the SPRIworks for the remainder of sample preparation. The downside of this kit is that the non-random nonamers cannot amplify all areas of the genome and certain portions of genes are often lost. Still, for many samples, this kit is the only way to generate RNAseq libraries.

NuGEN SPIA Chemistry. Image from NuGen.

Additional Chemistries Available in the BioMicro Center

Strand Specific Sequencing

For samples with high amounts of input, we can modify the chemistry of cDNA creation to allow detecting the strand of the RNA using the dUTP 2nd strand marking protocol that preformed best in J. Levin et al 2010. In this method, actinomycin D is added to the 1st strand synthesis to prevent reinitiation of the RT-PCR enzymes. Then, dUTP is substituted in for dTTP in the second strand, allowing a clear distinction between the forward and reverse strands. After library construction, but before amplification, the dUTP containing strand is degraded and only the reverse strand (template strand) remains. This method is highly efficient but requires significant amount of RNA as the actinomycin causes a significant reduction in 1st strand yield.

Sample Data from Strand Specific RNAseq

Size Selection

For some applications of RNAseq, such as splice choice determination, having a precise knowledge of the insert size is critical. While the SPRIworks does provide some size selection (typically restricting fragments to between 150 and 350bp), this can be too wide for some methodologies. In these cases, after libraries are amplified, they can be run on the Sage BluePippin (either singly or pooled). Here the size distribution can be much tighter, with most of the DNA fragments being within a 50nt range.

Comparison of the RNAseq methods

The BioMicro Center has done testing in head-to-head competitions of the TruSeq, NuGEN v2 and Clontech kits. These data were presented at AGBT 2012 as part of a poster. The authors were: Avanti Shrikumar, Zachary Banks, Manlin Luo, Ryan Sinapius, Paola Favaretto, Jessica Hurt, Chris Burge, and Stuart S. Levine. Selections of the poster are shown below:

Summary Sequencing of the transcriptome (RNAseq) has become an increasingly important tool in the molecular biology toolkit and is rapidly replacing microarrays as the primary method for determining genome-wide expression levels. Several vendors have created pre-packaged kits for creating RNAseq libraries for Illumina sequencing. These kits differ significantly in methodology and in the amounts of input required. Here we provide a head to head test of five different RNAseq kits in a core setting. The kits were evaluated on two experimental samples with similar expression patterns, murine embryonic stem cells and the same cells with a single factor knocked down by RNAi, to determine the sensitivity of each method. Each kit was additionally evaluated across three different concentrations of RNA input. We found that the different methodologies show different RPKM levels for each transcript and also vary in their technical reproducibility. The different methods resulted in small but largely distinct lists of differentially expressed genes that we compared to genes with known expression changes		Kit Type Home Made (Burge Lab) Illumina TruSeq NuGen v1 NuGen v2 Clontech SMARTer 1ug X X 100ng X X 10ng X* X X X 1ng X* X X 0.1ng X* X
	Experimental Design: ES cells were transfected with siRNA targeting a splicing factor or a control siRNA. The tested splicing factor normally blocks inclusion of specific exons that had been previously identified by RT-PCR. Reduction of the splicing factor’s levels should lead to an increase in the amounts of these specific transcripts. RNA was collected from the cells and analyzed by RNA-seq. Samples were sequenced to a depth of at least 7.5m reads of 40nt length on either a GAIIx or a HiSeq2000	Testing Matrix: A single sample of control and splicing factor knockdown samples were serially diluted. Aliquots of the diluted samples were tested against the 5 methods to determine the ability of each kit to identify differentially expressed genes in a biologically challenging situation as well as to identify their sensitivity to different input amounts and quantify the amount of technical variation. * indicates the amount tested was below the minimum recommended input.
Fraction of reads aligned: Reads aligning once or multiple times to the mouse genome (mm9) are shown. Reads were aligned with Bowtie. NuGen samples show an increase in non-aligned reads at low input amounts.	3’ Bias: Read densities were calculated along the exonic portions of each transcript. Transcripts were grouped by length and plotted as metagenes with both 5’ and 3’ ends locked. Clear 3’ bias can be observed in the samples processed using the Clontech kit.	Variation in RPKM score across the transcript.(right) The evenness of coverage within the transcript was measured by comparing the read density in largest two exons. Only exons greater than 200nt and genes with PRKMs over 10 were included in this analysis (n = 1,876 genes including 3,676 exons) (left) the average number of exons in the above data set with very low coverage (<5 reads) is shown.

RNAseq References

A few notable references. Please feel free to add more:

<biblio>

1. Paper1 pmid=18978772
2. Paper2 pmid=19015660
3. Paper3 pmid=18550803
4. Paper4 pmid=20711195

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