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7

The Next-Gen sequencers cannot sequence a very long stretch of DNA with good reliability (~150 for the recent model- HiSeq2000; even less for older models such as GA (40), GA-II (70), GA-IIx (90)). For increasing the confidence in a certain hit, it was sequenced from both the ends. For example, if you have selected 500bp DNA fragment, then after ligating ...


7

The MIT synthetic chemist Gobind Khorana won the 1968 Nobel Prize in Chemistry for his work which successfully was able to make chains of Ribonucleic acids. The chemistry was difficult at the time but he won the prize for making specific sequences of RNA bases which were then fed to cells, resulting in specific amino acid chains, which ultimately deciphered ...


6

RNA-seq data usually provides a snapshot in time of the transcriptome of that which is being sequenced. Single-cell sequencing is possible, but less common than RNA-seq on a sample (containing many cells). You are correct that RNA-seq provides one with knowledge of RNA sequences, like AUGGUCAUCAG and so on. However, one will not necessarily have ...


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): ...


5

There are some databases in which you can search for E.coli gene expression data: GenExpDB: E. coli Gene Expression Database Many Microbe Microarrays Database (M3D): A resource of microbial gene expression data Stanford MicroArray Database (use the search tool to find relevant organisms) Colombos (COLlection Of Microarrays for Bacterial OrganismS) ...


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 ...


4

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 ...


4

Good question, a lot of this is still being figured out. Here's what's known so far: Fragmentation methods based on restriction enzymes aren't random. Reverse-transcription performed with poly dT-oligomers, which bind to the 3' poly-A tails, is strongly biased towards 3’ end of transcripts. Reverse-transcription with random hexamers results in an ...


4

In Illumina sequencing, the DNA is (usually randomly) sheared into fragments. For paired end sequencing, fragments of a specific size range are selected and then sequenced from both sides. This results in two reads for each fragment. As read length is fixed, also the remaining "middle part" of the fragment is in a specific size range. In some cases there is ...


4

In RNA sequencing, the RNA is fragmented, DNA is synthesized complementary to the RNA fragments, which is followed by a complementary strand synthesis. Fragmentation can be done after the cDNA synthesis too. This DNA is then amplified to form a cluster that is sequenced. Most Next-Gen sequencing approaches sequence a short segment of the DNA (it has ...


3

I guess this arises because of the default cufflinks option --total-hits-norm in which it normalizes the FPKMs with total reads including the ones that are not mapped to a known gene or a predicted gene from the assembly. In the genes.fpkm_tracking file the FPKM values are reported for known/predicted genes. It is certainly possible that the number of genes ...


3

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, ...


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 ...


3

The process of sequencing was, and can be, assisted by cloning a DNA fragment into a known site in a plasmid designed to help sequencing. That cloning site is flanked by a known sequence(s) that can use standard primers In the figure "A" and "B" are just reference points on either side of the Cloning site. Of course, the plasmid extends on either side of ...


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

In RNA sequencing, total RNA is extracted, then mRNAs are purified out from the sample using polyT columns (since mRNAs have a polyA tail, this will attach to a polyT DNA chunk that is attached to some solid surface). This step is necessary because ribosomal RNAs are much more abundant than mRNAs, and for sequencing you only want to use mRNAs. Then the ...


3

Have you discovered wormbase yet? It will become your new best friend. In worms gene names reflect the loss-of-function (lf) phenotype. Daf means "abnormal DAuer Formation," so daf-2 was the second daf gene identified (ref). The mutations are called alleles and each worm lab has their own allele designation. e was for England and in that lab's strain list ...


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

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 ...


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.


2

Tour sequence's complementary is TAC CCC GGG AAA TTT ATT. Together they form a DNA strand. Now replicate this and then, after transcription of both the DNA you get the RNA sequence you wanted. Use it as sense or antisense.


2

Being a bioinformatician as well, I am not really what you asked for, but I work with plant genetics so I'll try and answer anyway. What you are mapping is RNA. So, as you already figured out, splicing events will be a problem for end-to-end mapping of the reads. There are tools managing that, though, so let's assume you used one of them and still a lot of ...


2

For the classic Sanger sequencing you need quite large amounts of DNA to be able to run the sequence reaction. This is a problem when you want to sequence whole genome since the DNA needs to be amplified then, which for some sequences can cause problems, introduce errors or cause bias. Single molecule sequencing is a huge step forward here, since only one ...


2

messenger RNA has a poli A tail at its 5´ end. thus when the poly-dT hybridize the mRNA in the reverse transcription the cDNA will carry this poly-dT at its 3`end. Thus, as reverse transcription will always start at the 3´ and of the mRNA its is more likely that this region is better rev. transcribed. Bigger the mRNA is bigger will be this concern. About ...


2

When you say RPKM do you mean crude RPKM or the estimates that you get using expectation maximization methods like cufflinks and eXpress? It is better if you get your RPKM or FPKM values from one of these programs because you can differentiate between transcript variants. I have mostly used cufflinks and eXpress. Cufflinks package is better for multiple ...


2

In my opinion you should try low input library prep protocols because in my experience even with a decent quantity of RNA, standardizing the sequencing is not an easy job. The manual says that this kit works with 10-20ng of RNA; always assume the higher limit to be true. There is a higher chance of loss (relative) during extraction also. Try doing ...



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