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We asked – When it comes to RNA-Seq library prep protocols, which parameters are the most important to you?

Thanks to all who participated in this poll!

As far as Next-Gen Sequencing application go, RNA-Seq is hot and has been getting over the last few years.  We thought it would be interesting to know for how long our readers have been interested and/or involved with RNA sequencing.  Check out our latest poll in the left-hand sidebar and cast your vote!

Incoming search terms:

• gljtjqaqh
• www rna-seqblog com rna-seq-blog-poll-results-12

Well, we let this poll go on a little bit longer than normal hoping that one of the choices would pull away from the other.  But alas, the split has been nearly equal since the poll has been up.  There have been many predictions on the Death of Microarrays but it seems they may be here to stay.

The future of microarrays?

Here to stay (i.e. Affy and Agilent affirm continued commitment) (50%, 88 Votes)

Its curtains (i.e. Roche shuts down Nimblegen, Combimatrix trading at $0.85) (50%, 87 Votes)

Total Voters: 175

Check out our latest poll in the left-hand sidebar and cast your vote.

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We asked: Do we yet have a firm handle on the most appropriate/accurate pipeline for analysis of RNA-Seq datasets?

The overwhelming result was: NO.

Given these results, we thought we should did a little deeper and find out what y’all think is the source of the problem.  Check out the latest poll in the right-hand sidebar and cast your vote.

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We’ve reached the magic number of 100 votes, so its time to retire the current poll and take a look at the results.

We asked – What sequencing depth is required for gene expression analysis? (i.e. Human sample, paired- end mappable reads, > 30 NT)

Results – Total Voters: 101

I think its interesting that the results do not fit to a bell curve.  Also, as noted by a blog reader, the results could be biased due to the recent release of Best Practices for RNA-Seq by The ENCODE Consortium which recommends 30M reads for “experiments whose purpose is to evaluate the similarity between the transcriptional profiles of two polyA+ samples” and 100M – 200M for “experiments from a typical mammalian tissue or in which sensitivity of detection is important”.

Please check out our new poll in the right-hand sidebar and cast your vote.

We asked the question: Which technology will ultimately rise above all others as the choice method for high-throughput sequencing?

A total of 74 RNA-Seq Blog readers voted.

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We asked: In the Year 2015, what will it cost to perform RNA-Seq (whole transcriptome sequencing – human sample)?

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• SEQanswers – RNA Sequencing

  • DESeq; can I omit timepoints during dispersal estimation? May 24, 2013
    I have a bacterial timecourse with 2 biological replicates per timepoint. There is a fair bit of variance between my replicates. I have spent the... […]
    amcloon
  • HT Seq Count stranded options May 24, 2013
    I am very new to bioinformatics, so I would be really grateful for some help! I have been using *HTSeq Count v0.5.3* and I am bit confused about... […]
    qwrissie
  • Tophat 2.0.8b installation error May 24, 2013
    I install tophat-2.0.8b to rerun the mapping. but when i make it, the error appears like this. make[1]: Entering directory... […]
    canhu
  • reason for low mapping rate?? May 23, 2013
    we did RNASeq using HiSeq 2000 100PE. When the data were back, I mapping them to the reference sequence, but got very low mapping rate (30-40%). I... […]
    miaom
  • cross-species data - questions about normalization May 23, 2013
    Hi, I have some data form various samples (cell types) in different species. I want to compare and analyze gene expression variability across the... […]
    trelek2
  • CuffDiff strange output May 23, 2013
    Hi, I hope that someone can be so gentle to help me. I'm analizing some data from RNA-Seq with TopHat and Cufflinks and I focus my attention on... […]
    Pruexel

• Biostar – RNA-Seq

  • Why am I getting so many unmapped reads in STAR, classified as "too short"?
    I am currently using STAR to map several Hi-SEQ mRNA runs. I'm having trouble getting a decent amount of reads to map, but I don't really understand why. I'm hoping you can shed some light :) In the final log, only about 50% (or less) of the reads map to the reference. I'm using a GTF in addition to the genome. The unmapped bin that most […]
  • What are the best practices for SNP identification in RNA seq transcriptome data
    I have 20 RICE RNA seq tranascriptome data hiseq 2000 platform paired end reads. I aligned fasta reads with BWA and remove PCR duplicates with PICARD. Later I call SNP with samtools using various parameters. I would like to clarify what parameters should I used while alinging to reference rice genome for looking SNP location 100 bp upstream and 250 bp downst […]
  • How do TopHat options -g , --supress-hits, and Bowtie options interplay?
    Hi, I am currently using TopHat2 to map RNA-seq runs. I think there have been some changes pertaining the -g option. Does anyone know how it works now? I used to think that setting -g would look for n alignments for a given read, report them [if top-scoring] and discard those reads that had more than g [top scoring] alignments. Now, the description sounds mo […]
  • What happened to -k in TopHat for multiple-mapping reads?
    Selecting -g n in tophat does not discard reads mapping more than n, but instead only reports n alignments for those out all all their TOP scoring alignments. I think there used to be an option -k that would allow one to discard reads that topped x alignments -- whatever happened to that? I only see -g in the tophat 2 manual, no reporting options like before […]
  • Does tophat use the library-type information for mapping, or just for the XS flag?
    When I specify library-type to TopHat, i.e., first-strand, second-strand, unstranded, TopHat appends a value + or - to the XS:A flag, which is useful for subsequent analyses, such as annotation. However, does this information actually influence the "mappability" of reads, or is this unaffected? My thinking is that the information would be considere […]
  • Purpose of Y-shaped adapters in Illumina Sequencing?
    Hi all, Y adapters different sequences to be annealed to the 5' and 3' ends of each molecule in a library. The arms of the Y are unique, and the middle part, connected to the DNA fragment, is complementary. What are the advantages of this? My take of this over having fully-complementary adapters (ADAPTER1 - - - - - ADAPTER1) is that: -Upon primer a […]