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I have HiSeq data from mice exposed to two conditions. I would like to answer the following question: "Is there a significant difference in mRNA transcript levels when comparing condition A to condition B?"

To answer this question, do I have to map my HiSeq reads to the reference genome to determine expression values or is it acceptable to use a small set of reference sequences of interest (approx. 50)? These particular sequences are not annotated on the reference genome. What are the downsides to not mapping to the genome?

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  • $\begingroup$ how many mice are in your groups? do I understand you correctly that you have 50 mice in your reference group? $\endgroup$
    – Vance L Albaugh
    Commented Jul 7, 2016 at 21:46
  • $\begingroup$ Seven in each group of mice. I would like to examine transcript levels for 50 mRNA fasta sequences. Essentially, can I "fish" for only these 50 sequences out of my Illumina Hi-Seq data without mapping to the mouse genome first? Will a lack of mapping to the genome falsely inflate what is identified as one of my sequences of interest? $\endgroup$
    – user24933
    Commented Jul 7, 2016 at 22:24
  • $\begingroup$ Does this help? biostars.org/p/75462 It's a post about aligning sequences using a reference genome without annotating. Or this article about de novo transcriptome assembly: genomebiology.biomedcentral.com/articles/10.1186/… $\endgroup$
    – Gaius Augustus
    Commented Jul 7, 2016 at 22:24
  • $\begingroup$ @VanceLAlbaugh Thank you for your thoughtful edit and clarifying questions $\endgroup$
    – user24933
    Commented Jul 8, 2016 at 0:06
  • $\begingroup$ @GaiusAugustus The biostars post is helpful, thank you. Based on that post it seems that what I am proposing is the "quick and dirty way to do basic gene expression experiments", albeit perhaps less excusable given that there is a great deal of information known about my organism versus that of the biostars post. I feel that de nevo assembly will not be necessary for my purposes but thank you for the reference. I will proceed both with and without the genome mapping to compare. $\endgroup$
    – user24933
    Commented Jul 8, 2016 at 0:54

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It's going to be hard for you to generate an RPKM or RPM value without % aligning to the genome, or the transcriptome. Your read counts will have no context.

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