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5

It may be worth checking out the paper "Comprehensive comparative analysis of strand-specific RNA sequencing methods". The most common techniques sequentially ligate different RNA adapters to the 5' and 3' ends of each RNA molecule prior to cDNA synthesis. You will end up with an RNA molecule that looks like this (where A and B are the two adapters): ...


4

I found the link to a commercial product by Evrogen here: http://www.evrogen.com/technologies/normalization.shtml They claim their method is compatible with nextgen sequencing platforms: cDNA normalization using duplex-specific nuclease (DSN) is a highly efficient approach that can be applied for normalization of full-length-enriched cDNA (Zhulidov ...


4

The only information you are missing is a way to identify the splice sites. There are many ways of doing what you need. The simplest, assuming you are sure of the origins of the mRNA, is to use a BLAST flavor, either plain BLASTn or, even better, BLAT, to compare your mRNA sequence to the genome of interest. BLAT really should be all you need if the mRNA ...


3

Basically, all 16S genes are highly conserved, i.e., they share much identical bases. This means one can bind the 16S gene piece (after DNA was cut) to a specific other piece of DNA, even if you don't know exactly the 16S gene bases. Everything else is discarded then. Now finally, using PCR the rest is amplified and sequenced. Sequencing 16S only is much ...


3

Yes, it was odd that they said that. The second protocol with the polyA based method had another purpose though, which was "examine the reproducibility of period-regulated genes in an independent genetic background". I would suspect this was its main purpose, and not so much to look at fragment bias, which has already been done in other papers. They didnt ...


3

There is something called GEO, which is maintained by the NIH and is a massive collection of data obtained from RNA seq, microarray, etc. experiments. One thing you can do is search for a paper that has done what you are looking for. The paper may have a GEO accession number, and you can use that number at the GEO website to find the data you want. You may ...


2

You should ask this question over at biostars.org -- and could use a bit more clarity. Do you actually have RNA-seq data that you want to use to find the exon/splicing structure of an mRNA? First step would be to use a splicing-aware aligner. A few free ones are: STAR TopHat GSNAP Let us know why that wouldn't do what you want when you re-ask this ...


2

Starting out with RNA data is great, since you already have fully spliced entities, despite being in a different dynamic regime. As the chromatin landscape itself is dynamic and High throughput data exploration has only begun in the last decade, consider the following tools and results with care... You may find the following tools helpful: Archalign , ...


2

The quick answer is that you are misinterpreting ESTs. EST stands for Expressed Sequence Tag. They are obtained by collecting and sequencing any mature (i.e. poly-A) mRNAs found in a living cell. Well, it was living until the experiment was performed at any rate :). The term EST is not applicable to sequences derived from genomic data. It only applies to ...


2

I would first recommend you ask this question biostar as the subject matter you are inquiring about is much more relevant there. That having been said, you have another option which is to use an aligner that soft clips 3' ends of reads specifically to account for adapter (or polyA, or whatever) contamination that might have flew under your radar. STAR is ...


2

q-value is the corrected p-value to account for multiple testing (i.e. you are testing thousands of genes). Those with q-value <0.05 are significant experiment-wise. Cuffdiff uses Benjamini-Hochberg correction to compute FDR (i.e. q-value). The calculation does not depend on the number of replicates it's based on the distribution of p-values those, yes, ...


2

I've done a functional annotation of a non-model, and used Blast2Go with fine results. Specifically, I took all of my proteins and queried again the NCBI non redundant (nr) protein database using mpiBLAST, then I analyzed this output using Blast2GO (B2G4PIPE) and a local B2G Database.


1

blast2go is on the verge of getting commercialized (as they have started selling PRO versions) and my previous experience was not so good with it. I used IntrProScan to associate GO terms with the transcripts, it ran long but it reported all the possible sequence features. To use these custom annotation was tricky for visualization, but thanks to BiNGO, I ...


1

The CATGTC sequence at the end of the poly A tail is an artefact of the method used in constructing the original cDNA library. According to https://www.ncbi.nlm.nih.gov/nucest/EE485195.1 this EST comes from a library constructed in the Clontech vector pDNR-LIB The Clontech SMART cDNA cloning system manuals are linked to from here and the general manual ...


1

Normally, when you need two unique adapters, say A & B, on either end of unknown insert sequences, cohesive-end ligation is difficult because the insert sequences are "unknown". So you have to do blunt-end adapter ligation, in a reaction containing Unknown Inserts + adapter A + adapter B. This can result in 3 possible versions of insert-ligated product: ...


1

Helicos stopped shipping reagents around 2010-2011, as they couldn't compete with Illumina. For now it seems that reverse transcription -> Illumina-sequenced cDNA is so much cheaper than any other option, that's the method that is dominant. Oxford Nanopore has discussed direct RNA profiling, but unfortunately their first product still hasn't shipped (for ...


1

My 2 cents: To answer your main question: Is our terminology becoming inconsistent due to the rapid improvements in sequencing technology, or am I missing or misinterpreting something? I think what you are witnessing (being subject to(?)) is, as you say, an inconsistency of terms due to the rapid improvements in the field. I put the caveat in that ...


1

As far as I know it'spossible to reverse transcribe a rRNA gene, you may not get the best yields though. The secondary / tertiary structures will be a problem, however, there are reverse transcriptase's commercially available that can handle secondary structure: Thermo-X™ Reverse Transcriptase from Invitrogen or Sensiscript™ from Qiagen.



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