Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Long peak length from ChIP-seq data

    Hi All,
    I am new to this forum. I have recently started using AB Solid V 2.0 for ChIP seq of a Drosophila TF. I used 500 bp avg Chromatin for pull down and for the library preparation it was brought down to ~100-200bp.

    We ran MACS with default parameters on the data. I was surprised to see that mean peak length for Test IP vs.Input was ~1.6 Kb! (Similar results for Test IP Vs. Mock IP) There were peaks as big as 10 Kb at times. Is this common? How can I get better resolution? Please help!

  • #2
    what kind of ChIP-seq data did you use? if it is the histone modification data, the long peaks would not be surprise.
    Xi Wang

    Comment


    • #3
      Dear Wang,
      Thanks for your reply. I didn't use histone modification ChIP-seq. The data is for a Drosophila transcription factor. Please let me know if you need more information.

      Comment


      • #4
        i didn't know much about the solid protocol for ChIP-seq. but i think the early stage software tools dealing with ChIP-seq data are for illumina data. check if the fragmentation of the solid protocol is the same as that of the illumina.
        Xi Wang

        Comment


        • #5
          Fragmentation of IP DNA (~500 bp) for Solid is done using Covaris S2 sonicator (water bath based, similar to Diagenode's bioruptor). I checked the size of the fragments on Lonza flesh gel after sonication and it was tightly distributed around 100-200 bp.

          Comment


          • #6
            The DNA fragmentation was carried out twice? I suggest that you could upload the raw reads and the MACS results to a genome browser to see how MACS works, and how MACS gives such long peaks.
            Xi Wang

            Comment


            • #7
              I would suggest upload peaks and the reads that are responsible for these peaks to any genome browser. Compare the results and the distribution of reads in the peaks region. This may give a better idea.

              Comment


              • #8
                Thanks a lot for your comments. I could upload the mapped reads on UCSC browser. I could see good enrichment around known targets but there is noise in the data from adjacent regions. The data was subtracted with input and I am waiting for Mock subtracted data. Any suggestions on reducing such noise so that I can find smaller peaks? If I fail to get smaller peaks than is it OK if I use only those motifs which are near the peak summit?

                Comment


                • #9
                  It is ok to look for motifs near the peak summit. but if there is no such consensus motif, you can extend the region to the flanking step by step, and do motif discovery again. To explain why no small peak, I am wondering if the TF of interest binds to the DNA sequence with its co-factors and the ChIP-ed DNA also contains the binding sites of the co-factors. Good luck!
                  Xi Wang

                  Comment


                  • #10
                    Chiper,

                    normally a size-selection is done prior to adaptor ligation and sequencing and one tries to keep fragments intact (i.e. no additional sequencing). The way your sample was treated means each fragment can give several reads, and reads from different strands will be less well separated. If your average size is 500 bp you may still have some enriched fragments that are several kb. One binding site could then give several "subpeaks" due to sonication or alignment bias. The resolution is not the same as the peak with though, the summits / peak max should still be what you use as a center for motif searches. You may find it difficult to separate close binding sites though.

                    Comment


                    • #11
                      Chiper,

                      If you see this paper at http://www.nature.com/nmeth/journal/...meth.1371.html, says three kinds of peaks, may give some idea about long peaks...

                      Classes of ChIP-seq signals. Consistent with previous ChIP-chip results, ChIP-seq tag enrichments or 'peaks' generated by typical experimental protocols can be classified into three major categories: punctate regions covering a few hundred base pairs or less; localized but broader regions of up to a few kilobases; and broad regions up to several hundred kilobases.

                      Also I will look for motif location in the binding sites...

                      Comment


                      • #12
                        Thanks a lot Chipper and Rao for useful comments and the review link. I will look at the data again and write if there are other issues.

                        I love this community

                        Comment


                        • #13
                          Chiper,
                          Another solution is starting from ChIP: fragmentize the cross-linked sample to <500bp directly. Invitrogen has launched a SOLiD ChIP-seq kit, SOLiD™ ChIP-Seq Kit with ChIP Magnet, Cat. No. 4449638. In their manual, there are fragmentation condition using either bioruptor or covaris to shear chromatin into ~200-300bp fragments (actual range will be broader than this). For this case, you don't need 2nd covaris shearing for library construction. And, you will get better peak resolution.

                          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
                          8 views
                          0 likes
                          Last Post seqadmin  
                          Started by seqadmin, Yesterday, 06:07 PM
                          0 responses
                          8 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