10

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 ...


9

The read length has absolutely nothing to do with what you are sequencing. It is a characteristic of the sequencing technology you use. NGS sequencing techniques typically produce this sort of short read you're seeing. The read length does not change because you are sequencing a longer molecule. You would still get ~250nt reads even if you were sequencing an ...


8

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 ...


8

You should really read about both these file formats. As swbarnes mentioned, FASTQ and GTF hold different kind of information. GTF stores the annotation of a reference sequence. For example a GTF for a genome sequence will have the information about the locations of features such as genes, transcripts, exons, start codon etc. FASTQ stores the sequence of a ...


7

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'-...


7

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) ...


6

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 ...


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

You will likely need a tool to "map" the reads on the reference genome. You may find such a reference genome, together with annotations, here: ftp://ussd-ftp.illumina.com/. Mapping tools such as bowtie2 or bwa take fastq files and reference genomes and output mapping results in a format called sam. You then have a lot of options to estimate gene expression....


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

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

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

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

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 ...


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 under-...


4

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 ...


4

The frequency of the bases in the genome isn't equal to 0.25, the frequency depends on what kind of organism you mean. However let's take a look at some of them: bacteria, most of the time we can see a bias towards some bases, this could be a GC bias, for example if the bacteria lives in extreme conditions, because GC can vorm three hydrogen bonds compared ...


4

Q: Why not just extract the proteins… A: It’s not just a question of extracting the proteins, you would need to separate them and then isolate each of them. There is currently no practical way to do this for say, 10,000 proteins, which in any case may have multiple forms. The beauty of the RNAseq method is that it does not require physical separation or ...


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

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

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

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 ...


3

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 ...


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

A fastq contains sequences. A gtf contains coordinates of where features like exons fall in a reference sequence. You can't interconvert them, that makes no sense.


3

Your question is not very clear. What should the contents of your GTF file be? Typically, GTF files contain information about where exons are located in a set of DNA sequences. Determining the location and exon/intron structure of genes is no simple technical task (i.e. a "conversion" as stated in your question), but rather a large area of active research. ...


3

The Tuxedo Suite (Tophat, Bowtie and cufflinks) used to to process RNA_seq data, assuming that is the origin of your .fastq files, should work for you. https://ccb.jhu.edu/software/tophat/index.shtml


3

If you google HG00105, among the first hits is geo accession GSM649517 with the title HG00105/NA12878. Channel1: Characteristics gender: Male cell line: lymphoblast cell line HG00105 ethnicity: British from England and Scotland, UK (1000 Genomes codes: GBR) Channel2: gender: female cell line: lymphoblast cell line NA12878 ethnicity: Northwest European ...


3

It is difficult to draw any conclusion without further experimentation. There may be many other factors that prevent the expression of the gene including factors like post-transcriptional regulators. Some histone modifications like the ones you are mentioning are also a bit dicey and there can be bivalent modifications too. However, if there is a strong ...


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