Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • RNA-seq with paired-end reads

    anyone's doing RNA-seq with paired-end reads now? how to use pairing information is a challenge. any good ideas?

  • #2
    Yes. We've done paired end RNAseq - partly because of mixed sample type (some DNAseq etc) flow cells.

    The paired end info is useful for novel gene expression and splice junction discovery.

    For simple RPKM etc we just treated each read as single end and aligned to genome+splice junctions seperately - this is likely to be suboptimal although easy approach. I'd be very interested in people's comments...

    david

    Comment


    • #3
      Rna-pet

      Paired-end ditags (PET) is an interesting strategy. It is very economical way of finding splice junction variants. Of course it doesn't offer the same resolution as full paired end RNA-Seq. I believe the team at GIS have migrated to Illumina and SOLiD in particular for RNA-PET now.

      Comment


      • #4
        PE and long read would be better to detect splicing junction and SNPs.

        Comment


        • #5
          Hi all, We have had success with PE RNAseq (illumina).

          Some of the main pitfalls that we found:
          -when you make your cDNA, if you use random hexamers (best results for us), your first strand synthesis reaction will tend to produce sequences that start/end in GC rich sequences. We have found that these tend to be non-accurate annealling products, and you can improve your mapping efficiency by stripping the first/last few bases.
          - TopHAT (see bowtie/ben langmeads posts) has also been very useful (and extremely fast for mapping!!
          - Creating 'spliced' reference genomes is very useful for retaining the benefits of PE mapping, without having to introduce huge 'insert' sizes...
          - Avoid PET tags - the extra ligation steps, if you are not careful, can introduce a high percentage of 'false' splice events... With the illumina PE strategy there is no need for this...

          Has anyone had any luck with barcoding RNAseq samples?

          Ieuan

          Comment


          • #6
            Originally posted by ieuanclay View Post
            Hi all, We have had success with PE RNAseq (illumina).

            Some of the main pitfalls that we found:
            -when you make your cDNA, if you use random hexamers (best results for us), your first strand synthesis reaction will tend to produce sequences that start/end in GC rich sequences. We have found that these tend to be non-accurate annealling products, and you can improve your mapping efficiency by stripping the first/last few bases.
            - TopHAT (see bowtie/ben langmeads posts) has also been very useful (and extremely fast for mapping!!
            - Creating 'spliced' reference genomes is very useful for retaining the benefits of PE mapping, without having to introduce huge 'insert' sizes...
            - Avoid PET tags - the extra ligation steps, if you are not careful, can introduce a high percentage of 'false' splice events... With the illumina PE strategy there is no need for this...

            Has anyone had any luck with barcoding RNAseq samples?

            Ieuan

            Hi Ieuan,

            It is interesting to see PE RNAseq.
            I still ahve some doubts regarding mapping PE reads. Because ELAND can support eland_rna analysis for SR(single read) only.
            Any tool to map tags with splice juction or can you please explain about the mapping part of PE RNA seq tags?

            Thanks.

            Comment


            • #7
              Check the forums here for TopHat if you want to know about mapping RNAseq!

              The actual way that you map is very dependent on the source of your RNA, so i would need to know more about your experiments! My main experience is with nascent RNA, so we PE map againstt a genomic reference. Spliced RNA can be mapped against spliced genomes (again see TopHat). Or a combination of both can be used, i.e. mapping against the genomic reference using a sensible PE insert limit, to find nascent RNA, then mapping the reads that don't map to the reference genome against a spliced genome. This way you can flag potentially spliced and unspliced transcripts.

              There really are too many ways of proceeding, you just have to pick a processing pipeline that makes sense for your material/experiment and that you can justify when it comes to publishing.

              Hope this helps!?

              Comment


              • #8
                no one show how to use paired-end info to infer junction and alternative splicing?

                Comment


                • #9
                  hai ....I would like to know is it possible to use Bowtie to map the reads which does not have its genome sequenced?

                  Comment


                  • #10
                    You can't use Bowtie without a reference genome. But there are others that can (Velvet, etc.)... just search for them in this forum...

                    Comment

                    Latest Articles

                    Collapse

                    • seqadmin
                      Strategies for Sequencing Challenging Samples
                      by seqadmin


                      Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
                      03-22-2024, 06:39 AM
                    • seqadmin
                      Techniques and Challenges in Conservation Genomics
                      by seqadmin



                      The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                      Avian Conservation
                      Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                      03-08-2024, 10:41 AM

                    ad_right_rmr

                    Collapse

                    News

                    Collapse

                    Topics Statistics Last Post
                    Started by seqadmin, Yesterday, 06:37 PM
                    0 responses
                    11 views
                    0 likes
                    Last Post seqadmin  
                    Started by seqadmin, Yesterday, 06:07 PM
                    0 responses
                    10 views
                    0 likes
                    Last Post seqadmin  
                    Started by seqadmin, 03-22-2024, 10:03 AM
                    0 responses
                    51 views
                    0 likes
                    Last Post seqadmin  
                    Started by seqadmin, 03-21-2024, 07:32 AM
                    0 responses
                    68 views
                    0 likes
                    Last Post seqadmin  
                    Working...
                    X