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  • Solexa-mRNAseq

    Hi everyone,

    I am using Illumina mRNAseq kit to prepare mRNA library for sequencing. I have two questions:
    1- when you cut the band from gel after PE adaptor ligation, what range is best to cut? Illumina says "200bp" band, our core facility scientist says "200-400bp" and I personally think 190-300bp should be the best. My argument is based on the mRNA fragments produced after the fragmentation step and the length of the adapters. Fragmentation produces 60-200bp fragments (see Ambion fragmentation buffer notes) and the total length of the PE adapters is 120. This will make a range of 180-320. Is this make sense, anyone agrees with me?

    2. I use the Sera-mag oligo dt particles to purify the mRNA from total RNA in two rounds of hybridization and elution. Accidentally, I made the first elution round in 1 M tris-Hcl (pH 7.4) instead of 10 mM of the same buffer. I corrected this in the 2nd elution round. I then used Nanodrop to quantify the obtained mRNA. It was around 5 ng/ul....Sound normal. Does anyone experienced this issue before. I mean should I proceed with this mRNA or not? I am going to test it with Agilent bioanlyzer, but I am still confused whether I use it or start all over again. Any advise.

  • #2
    I can't really advise on #2, but I strongly suggest that you cut relatively high and narrow. If you have a good control over your insert size, the paired-endedness will be more informative.

    Ali

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    • #3
      Yo M,

      Long time no see. As far as your question, I'd agree with high and narrow as long as you can get enough DNA. Depending on your read length, too short of an insert length might set you up for reads that overlap (even if it's only a small percent).

      Brad

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      • #4
        Hola,

        I see now. for my first question, it is clear that most of the fragments after adapter ligation will be in the 200-250 range. And this what I cut. I got nice results.
        For my second question, it works just fine. If anyone done this mistake, go ahead and elute again in water or 10mM tris HCl. 1M tris-HCl will not affect the bead plus temperature is the most important factor at this step.

        Hola Brad, nice to see you. Once again, we are doing similar stuff.

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        • #5
          It is probably a good idea to cut multiple bands as separate samples for PCR amplification. If you want to go back and run PE mRNAseq then a range of insert sizes would be best.
          PE mRNAseq should return data where 95% of read-pairs give information on splicing rather than the 3-5% of reads from SE mRNAseq.
          It is simple to do this and you can just run whatever you are hapy with today in the first instance. I cut 200,300,400,500 and 800bp products and they all PCR amplified reasonably well. I have not sequenced the other size bands but the 30bp range worked great.

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          • #6
            Hi,
            I obtained the small RNA sequences for library prepared using the new protocol (V1.5). What is the best way to trim the 3' adapter. since the small RNA sequences are of different lengths, the sequenced portion of the adapter will be of different lengths as well, so it is not straight forward way to trim adapters. ANY HELP

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            • #7
              I assume you will be aligning these reads to a reference. For aligning small RNAs (e.g miRNA) I have used Novoalign (www.novocraft.com) which works well for two reasons. First, if you are looking for miRNAs it has a scoring function specific for miRNAs and second, it can recognize and trim the Illumina adapter from the 3' end of the read automatically. While Novoalign is a commercial piece of software it is made available free for personal, non-commercial use.

              If you want to trim the adapters yourself I would suggest fuzznuc which is part of the EMBOSS package. With it you can define a sequence including ambiguities (not required in your case), allow for mismatches (definitely helpful) and anchor the search to one end of the read (the 3' end in this case).

              You will end up with reads of varying lengths. Many of the current short read aligners don't work with variable length reads so you would have to deal with that.

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              • #8
                Originally posted by james hadfield View Post
                It is probably a good idea to cut multiple bands as separate samples for PCR amplification. If you want to go back and run PE mRNAseq then a range of insert sizes would be best.
                PE mRNAseq should return data where 95% of read-pairs give information on splicing rather than the 3-5% of reads from SE mRNAseq.
                It is simple to do this and you can just run whatever you are hapy with today in the first instance. I cut 200,300,400,500 and 800bp products and they all PCR amplified reasonably well. I have not sequenced the other size bands but the 30bp range worked great.

                Do you run the different bands as separate samples or do you pool them back together again for sequencing? I'm planning on trying a modified version of the NlaIII tag digital gene expression where I would expect insert sizes of 15-250 plus 120 bp for PE adapters (?) for a total of 130-370 bp. Also, did you mean 300bp rather than 30bp, or am I missing something.

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                • #9
                  I don't think you want to pool them back since it makes a big difference to know the approximate distance between paired reads when aligning or assembling.
                  If you pool them you loose that advantage.

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                  • #10
                    I would not pool, however you could barcode and run a mix of inserts that would be most informative. What this mix might be would be difficult to guess at but ist is probabaly ~70% 'normal' and 30% longer inserts. The same principle might apply to structural variation?
                    Yes, 300bp.

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