These reads must then be aligned back to the reference genome. Early RNA-seq techniques used Sanger sequencing technology, a technique that although innovative at the time was also low-throughput and costly.
An RNA-seq workflow has several steps, which can be broadly summarized as:. The cDNA is then fragmented, and adapters are added to each end of the fragments. These adapters contain functional elements which permit sequencing, for example, the amplification element which facilitates clonal amplification of the fragments and the primary sequencing priming site.
Following processes of amplification, size selection, clean-up and quality checking, the cDNA library is then analyzed by NGS, producing short sequences that correspond to all or part of the fragment from which it was derived.
The depth to which the library is sequenced varies depending on the purpose for which the output data will be used for. Sequencing may follow either single-end or paired-end sequencing methods. Paired-end methods sequence from both ends and are therefore more expensive 6 , 7 but offer advantages in post-sequencing data reconstruction. A further choice must be made between strand-specific and non-strand-specific protocols.
The former method means the information about which DNA strand was transcribed is retained. The value of extra information obtained from strand-specific protocols make them the favorable option.
These reads, of which there will be many millions by the end of the workflow, can then be aligned to a reference genome if available or assembled de novo to produce an RNA sequence map that spans the transcriptome.
RNA-seq is widely regarded as superior to other technologies, such as microarray hybridization. Not limited to genomic sequences — unlike hybridization-based approaches, which may require species-specific probes, RNA-seq can detect transcripts from organisms with previously undetermined genomic sequences. This makes it fundamentally superior for the detection of novel transcripts, SNPs or other alterations. Low background signal — the cDNA sequences used in RNA-seq can be mapped to targeted regions on the genome, which makes it easy to remove experimental noise.
Furthermore, issues with cross-hybridization or sub-standard hybridization, which can plague microarray experiments, are not an issue in RNA-seq experiments. More quantifiable - Microarray data is only ever displayed as values relative to other signals detected on the array, whilst RNA-seq data is quantifiable.
RNA-seq also avoids the issues microarrays have in detecting very high or very low transcription levels. Propelling Progress with RNA-Seq RNA sequencing can have far-reaching effects on research and innovation, transforming our understanding of the world around us. Benefits of RNA Sequencing.
Download eBook. Access PDF. New To NGS? Find out how Illumina NGS technology works and what types of experiments it enables. Microarrays RNA-Seq vs. Learn More Ribosome Profiling Deeply sequence ribosome-protected mRNA fragments to gain a complete view of the ribosomes active in a cell at a specific time point, and predict protein abundance.
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Read Interview. Read Technical Bulletin. The flow cell is a glass slide with lanes coated in a lawn of the two different types of oligos complementary to our adaptor sequences. This allows for our transcripts to transiently bind to the flow cells for sequencing. The second function of adaptors is to serve as priming sites for the polymerases used in sequencing. After adaptors are ligated to the cDNA molecules, many library preparations undergo a process of indexing.
This barcode allows for the transcripts to be identified during the sequencing process after pooling samples. Pooling is a process that involves mixing numerous different samples together at a known concentration so they can be added to the flow cell and sequenced simultaneously.
Pooling samples is often done to save time and money. After adaptor ligation and indexing, samples are ready for sequencing! Step 1 the process of adaptor ligation and indexing involves the addition of the synthetic oligonucleotides to our target cDNA molecules. Step 2 an adaptor, with the unique barcode is ligated to the cDNA target. Illumina adaptors are commonly used, they are designated P5 and P7.
Step 3 the other adaptor is added to the other end of the cDNA molecule. These amplified libraries are then quantified to determine their concentration. The concentration of the libraries is then normalized to ensure the libraries are sequenced evenly and that no one library is overrepresented during the sequencing process.
There are a few different technologies for sequencing such as sanger sequencing, and more high throughput options such as pyrosequencing, ion torrent, and nanopore sequencing. There are two parts of sequencing by synthesis, which are cluster generation followed by the actual sequencing process.
Our samples are now each indexed, meaning they have a unique barcode tag that allows us to identify the samples after multiple samples are pooled together. The pooled samples are added to a flow cell in the sequencer. Step 1 the adaptorized transcripts can hybridize to the complementary oligos of the lawn so they are bound to the flow cell. The flow cell oligo serves as the primer for a polymerase to create a complement of the hybridized fragment. Then the double stranded molecule is denatured, and the original template is washed away leaving only the newly synthesized strand that is bound directly to the flow cell.
Step 2 the strand now folds over, and the adaptor region hybridizes to the other kind of oligo on the flow cell and a polymerase uses the new oligo as a primer to create a complementary strand again. Step 3 now there is a double stranded bridge of complementary strands. Step 4 the bridge is then denatured resulting in 2 single-stranded copies of the transcript, both bound to the flow cell.
Step 5 this process is called bridge amplification and it is repeated many times resulting in the generation of many copies of the same molecules across the flow cell, these are the clusters. Now that we have generated clusters, the reverse strands are cleaved and washed away. This leaves forward strands to begin sequencing. Sequencing begins with extension of the first sequencing primer to produce read 1, or the forward read.
Step 1 fluorescently tagged complementary nucleotides are added to the chain one base at a time based on the sequence of the template.
Each nucleotide is tagged with a different color fluorescent signal. Each nucleotide is also a reversible terminator, meaning that after it is incorporated into the chain, another cannot be added.
Step 2 after the nucleotide is added to the chain, a light source excites the clusters and a fluorescent signal is emitted and read by the sequencing machine. The emission wavelength allows the computer to determine which base was added to the chain, which is a base call. The intensity of the signal produced will determine the confidence score for the accuracy of the base call.
Step 3 after making the call, the reversible terminator is cleaved, and the chain is ready for the addition of the next nucleotide. If you are interested in simple differential gene expression, then strand information will not add much to your experiment, but will make your protocol more complex.
Having said that, you can perform the most widely adopted method without too much extra effort. To do this, during 2 nd strand cDNA synthesis, use uracil for incorporation instead of thymine. Follow the Illumina library prep as normal, but after adapter ligation and before PCR amplification add uracil-DNA glycosylase to degrade the 2 nd strand. This results in all reads starting in the same orientation so you can determine which strand was being transcribed in your sample.
RNA-seq is a powerful and versatile tool published widely over the last few years. I have picked a couple of my favorites some from work performed in the core facility I manage to illustrate what you can do with RNA-sequencing. In summary, RNA-seq is still an evolving tool, but is preferable in most instances to microarrays.
It is more sensitive, more robust and can be more cost effective. What RNA-seq projects are you now planning for your project? Journal of Investigative Dermatology Nature Genetics Levin et al : Comprehensive comparative analysis of strand-specific RNA sequencing methods.
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