Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Building a genetic map from RAD-seq using OneMap

    Dear all,

    I have some RAD-seq data from 2 parents and 97 offsprings. I would like to use this data to cluster my genomic scaffolds into linkage groups. I'm new to handling RAD-seq data and I'm not sure if what I'm doing is correct.

    For the moment I have done the following:
    - Quality Control of the RAD-seq reads
    - Demultiplexed the files
    - Mapped the RAD-seq reads to the genomic scaffolds (Bowtie2 + Samtools)
    - Ran the ref_map.pl pipeline of Stacks, specifying a "onemap" output from the program "genotypes"
    - Followed the tutorial step of OneMap (https://cran.r-project.org/web/packa...ed_version.pdf)

    Now here is where things start not working: as I increase the LOD value, I get more linkage groups but I also increase the number of unlinked markers.

    For example, with LOD=6, I get:
    > groups
    This is an object of class 'group'
    It was generated from the object "all.mark"

    Criteria used to assign markers to groups:
    LOD = 6 , Maximum recombination fraction = 0.5

    No. markers: 1342
    No. groups: 1
    No. linked markers: 901
    No. unlinked markers: 441

    With LOD=20, I get:
    > groups
    This is an object of class 'group'
    It was generated from the object "all.mark"

    Criteria used to assign markers to groups:
    LOD = 20 , Maximum recombination fraction = 0.5

    No. markers: 1342
    No. groups: 9
    No. linked markers: 225
    No. unlinked markers: 1117

    Is it normal to lose so many markers? What am I doing wrong?

    Also some groups (with LOD=20), only have 2 markers which I guess is insufficient to have a good genetic map.

    Any help or insight about building a genetic map using RAD-seq highly appreciated!

    Cheers!

  • #2
    You could write your question to the Stacks Google group (https://groups.google.com/forum/#!forum/stacks-users). Julian is pretty responsive.

    I'd say it is normal to increase the number of unlinked markers as you increase the LOD score stringency. I don't know if your numbers look typical though.
    Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com

    Comment


    • #3
      Thanks for answering! I'll follow your advice and post my question on the Stacks forum. Thanks for the link!

      Comment


      • #4
        Hi standonn,
        I have the same problem. Do you have a solution now?

        Comment


        • #5
          Yes, the problem was solved the following way:

          First, after demultiplexing the RAD-seq data, I ran the denovo_map of stacks. I specified "F2" as the cross (option -A). Depending on the cross that was performed choose the one corresponding to your data. I think this was my problem. I didn't choose the right cross thinking I had another type of data. Once that confusion was sorted, everything started to work.

          After running deno_map.pl I ran the genotypes program the following way:
          genotypes -b 1 -P . -r 7 -t F2 -o onemap -c

          This allowed me to get the genotypes for each marker and of each progeny sample in the onemap format.

          Then I ran onemap to build linkage groups and order the markers:
          This is what I got:
          > F2 <- read.mapmaker(file="for-onemap-analysis.txt")
          --Read the following data:
          Type of cross: f2
          Number of individuals: 95
          Number of markers: 1329
          > twopts.f2 <- rf.2pts(F2)

          > mark.all <- make.seq(twopts.f2, "all")
          > (LGs <- group(mark.all, LOD=20, max.rf=0.5))
          This is an object of class 'group'
          It was generated from the object "mark.all"

          Criteria used to assign markers to groups:
          LOD = 20 , Maximum recombination fraction = 0.5

          No. markers: 1329
          No. groups: 7
          No. linked markers: 1294
          No. unlinked markers: 35

          With this LOD/max.rf I built 7 groups (and I know this is the number of chromosome of my species). These groups contain most of the markers. I then continued the analysis: ordered the markers using the RECORD method, cleaned the map using R/QTL (with R/QTL you can remove duplicate markers and samples, remove markers/samples with a lot of missing data, look at the segregation distortion...).

          I hope this helps. Ah, I also re-ran the stacks ref_map.pl pipeline and it gave me the same result (the maps from the denovo_map.pl and from ref_map.pl are extremely similar).

          Best,
          Sophie

          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
          10 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, Yesterday, 06:07 PM
          0 responses
          9 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          49 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          67 views
          0 likes
          Last Post seqadmin  
          Working...
          X