Read Aligner Rnaseq to Genome 75 Paired End

Genome-Wide RNAseq and Assortment-Based NanoString Transcriptomic Technologies: Which To Use and When?

by , September 10, 2022 at 12:00 PM

RNA strand for use in transcriptomic technologies Compare the pros and cons of 2 widely used transcriptomic technologies - genome-wide RNAseq and array-based NanoString.

The Rise of Transcriptomics in Oncology

The rapid expansion of precision oncology approaches in recent years has been fueled in part by technological innovations and an increased interest in personalized therapies. Precision medicine approaches not merely rely on biomarkers to define optimal treatment strategies but also play of import roles in the diagnosis of illness, treatment efficacy assessment, and/or disease progression.

Cistron expression studies are a useful tool to identify biomarkers that are used to diagnose cancer and predict treatment efficacy and disease prognosis. The recent explosion of loftier-throughput technologies that generate big-scale molecular measurements has accelerated gene expression biomarker evolution. In detail, transcriptomics is a powerful arroyo for biomarker discovery because information technology profiles the entire set of RNA transcripts produced past a genome.

This post helps yous choose the right transcriptomic applied science for your mRNA profiling studies, focusing on the master steps, advantages, and disadvantages of RNAseq and the NanoString platform, with a brusk review of other available technologies.

RNA Sequencing (RNAseq)

RNA sequencing, or RNAseq every bit it is more commonly known, direct sequences and quantifies the number of mRNA molecules in the entire transcriptome. This technique is relevant to a wide range of applications, including identifying changes in gene expression associated with a specific affliction state. For instance, profiling cancers with RNAseq provides key insights regarding tumor classification and progression past identifying variants expressed in unlike samples.

The Main Stages of RNAseq

In that location are iv main steps in RNAseq which cover:

mRNA transcript fragmentation, followed by random primer binding to mRNA segments
Generation of double-stranded DNA from showtime- and second-strand cDNA synthesis via contrary transcription of the mRNA fragment
Tagging of the ends of the fragments with a phosphate grouping and poly(A) tail
Ligation of adaptors onto the cDNA fragments, which enables downstream PCR amplification and sequencing.

It'southward key to understand the main advantages and disadvantages of RNAseq, to determine if information technology'southward the optimal assay for your specific inquiry needs.

Advantages of RNAseq

Different traditional hybridization-based technologies (e.g. arrays), RNAseq does not require specific probes (i.due east. species- or transcript-specific probes). This makes it a powerful tool for detecting novel transcripts, cistron fusions, unmarried nucleotide variants, indels (pocket-sized insertions and deletions), and other changes that arrays are not able to detect.
Genome-broad profiling technique that can assess the approximately 26,000 genes in the man genome, rendering it a powerful tool for biomarker discovery.
RNAseq is highly sensitive and can be used to detect rare and low-abundance transcripts (e.1000. weakly expressed genes, unmarried transcripts in a cell) by but increasing the depth of sequencing coverage.
It enables discovery of novel circuitous genomic features, such as previously unknown transcriptional features, including alternative splicing, allele-specific expression, and newly transcribed regions.

Disadvantages of RNAseq

Loftier-quality RNA is needed, with a suggested RNA integrity number over viii.
RNAseq as well requires a minimum of 1µg total RNA, simply ≥2µg is preferred, which are larger samples compared with NanoString.
Consummate removal of genomic Dna is cardinal. Therefore, DNase handling is necessary because amplified RNA cannot be differentiated from genomic Deoxyribonucleic acid.
Downstream analyses require experienced scientists to leverage the data to its full chapters. Processing raw data to obtain normalized gene expression tin can also be costly.

NanoString

NanoString Technologies, Inc. developed a technology known as "nCounter," a variation on Deoxyribonucleic acid microarrays, that is robust, sensitive, reproducible, piece of cake to employ, and amplification free. The NanoString platform counts individual mRNA transcripts. It's useful for many potential applications, such every bit studying gene regulatory pathways, diagnostics, validating gene expression experiments in disease studies, including cancer, and ultimately generating highly translatable data for clinical utilize.

The Main Steps using the NanoString Platform

NanoString uses 2 hybridization probes (each ~35–50 bases in length) per mRNA molecule. Probe pairs are generated for each unique gene. The reporter probe carries the signal. The other probe "captures" the complex, immobilizing it so that a digital analyzer tin detect the hybridized reporter probes (there are four different-colored fluorophores). The probes are mixed with the RNA in solution. The digital analyzer identifies, scans, and counts the molecular barcode per sample. Notably, since the probes amalgamate directly to the RNA, a reverse transcription step is not required to generate cDNA.

Advantages of NanoString

High-quality RNA is not required. Information technology is therefore ideal for scenarios where only poor-quality RNA is available, including formalin-fixed paraffin-embedded material.
Smaller amounts of starting material are needed compared to RNAseq: 100ng of total RNA or lysate from 10,000 cells.
There's minimal background betoken.
It is amplification gratuitous.
Reduced possibility for introducing bias.
The NanoString platform has improved detection of low-expression RNA.
Data assay software is freely provided by NanoString Technologies, with readouts that are user friendly and suitable to nonspecialists.

Disadvantages of NanoString

It is not suitable for biomarker discovery, since it employs approximately 800 preselected genes that are publication based.
Information technology is a proprietary platform that offers limited flexibility.

Comparison of Assay Properties for Illumina MiSeq RNAseq, NanoString nCounter, and Deoxyribonucleic acid Microarray

Adjusted from Narrandes and Xu. Gene Expression Detection Assay for Cancer Clinical Utilize. J Cancer. 2018;9(13):2249-2265.

	Illumina MiSeq RNAseq	NanoString nCounter	Deoxyribonucleic acid Microarray
Primer/probe design	Primers on flow cell and adaptors to ligate to ends of sample	Capture probe with 3' analogousness tag and reporter probe with color-coded tag	Deoxyribonucleic acid oligo probes complementary to cDNA samples
Sample preparation	RNA extraction; reverse transcribed sample; fragmentation, library structure	RNA extraction	RNA extraction; reverse transcribed sample, fragmentation
Instrument	MiSeq benchtop sequencer	Prep station and digital analyzer	Microarray scanner
Reproducible	Yep	Yes	Yes
Specificity	Rely on data analysis	Design of capture and reporter probes	Density of probes annealed to the slide, probe pattern
Sensitivity	<1 copy/prison cell	<1 copy/cell	1–10 copies/prison cell
Clinical use?	Yep	Aye	Yes
Commercialized for clinical use	No	Prosigna (NanoString Technologies, Inc.)	MammaPrint
Number of genes or transcripts detected	Whole transcriptome	~800	50,000
Upward to sample # per assay	96	12	i–12/assortment
Processing steps	cDNA lib prep, sequencing, data assay	Label probes, hybridization to array, Information assay	Characterization cDNAs, hybridization to array, Data assay
Raw information assay	Past automobile in 3 hours	Past auto in 2.7 hours	By machine in 1 hour 40 minutes
Normalization	Reads per kilobase meg	Housekeeping genes; positive controls	Housekeeping genes; robust multi-array average; LOWESS method
Data analysis	Data output equally sequenced reads with quality scores or read alignments	Color-coded images are taken and output every bit code counts	Visualization; statistical tests

Other Available mRNA Profiling Platforms

Several other sequencing approaches can be used for mRNA profiling, which are briefly summarized here including a listing of some of the bachelor commercial products.

three'RNA Sequencing:

Hither, mRNAs are not fragmented before reverse transcription, and cDNAs are reverse transcribed only from the 3′ end of the mRNAs. Therefore, only one copy of cDNA is generated for each transcript. This ways that when the cDNAs are sequenced, the number of reads is a direct reflection of the number of transcripts of a specific gene.

Commercial Products: Lexogen QuantSeq, QioSeq UPX 3' Transcriptome

Specific Targeted Sequencing:

This approach targets RNA sequences by hybridization to DNA oligos followed past removing the unhybridized oligos and amplifying the remaining production.

Commercial Products: BioSpyder TempO-Seq, Ion Ampliseq Transcriptome

Traditional Microarrays:

These are used to quantify a divers gear up of transcripts by hybridizing the RNA to an array of complementary probes (which typically stand for to the 3′ stop of the factor). This technology can be used to evaluate thousands of transcripts simultaneously. Advances in fluorescence detection accept increased the power to accurately detect low-abundance transcripts.

Commercial Products: Affymetrix U133A, Illlumina BeadChip HT-12 v3/v4

Newer Microarrays:

These arrays focus on 20,000+ well-annotated genes and provide a gene-level view of the transcriptome. These newer arrays consist of divers sets of probes that target feature exons rather than the 3' stop of a gene.

Commercial Product: Affymetrix Clariom S

Conclusion

Transcriptomic data can be reliably generated from different measurement platforms, with the relative merits and limitations of the RNAseq and NanoString factor expression profiling platforms summarized here. Ultimately, the choice of platform used will depend upon the study endpoints, required resolution and coverage, throughput, sample quality, availability, and budget.

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Source: https://blog.crownbio.com/genome-wide-rnaseq-and-array-based-nanostring-transcriptomic-technologies