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  • adapters sequence fasta file

    hi there, do you have the sequence of adapters used in Illumina for library prep. so I can trim from the sequence data, please. thanks

  • #2
    See this thread for hints: http://seqanswers.com/forums/showthread.php?t=198

    Also here: http://support.illumina.com/download...es_letter.ilmn
    Last edited by GenoMax; 12-03-2013, 04:15 AM.

    Comment


    • #3
      For the most part you don't need to know the full adapter sequence for the specific adapters you used. Nearly all illumina adapters start with a common sequence and then later diverge into the different variants. If you trim based on the common sequence you will remove any instances of any of the other adapter types. The common sequence (as it would be seen in read-though) is AGATCGGAAGAGC.

      The only types of library we've seen where we get other adapter starts are in small RNA libraries where we run a trimming using ATGGAATTCTCG.

      Comment


      • #4
        Gff

        how to get GFF file for a certain genome (DNA)?

        Comment


        • #5
          Originally posted by mmmm View Post
          how to get GFF file for a certain genome (DNA)?
          Please start a new thread for questions that pertain to a new topic.

          Do this to start a new thread (always a good idea to search the forum first):

          1. Go to Seqanswers.com main page
          2. Choose "Forums" from "site navigation menu" (left side).
          3. Choose an appropriate forum for your post
          4. On the subsequent forum page use the "New Thread" button at top left of the page.

          That said, a GFF file may not be available for all genomes (you may need to construct one yourself). If you work with a model/common organism then you can get the files from Ensembl (http://useast.ensembl.org/info/data/ftp/index.html), UCSC or NCBI.

          Comment


          • #6
            I have a question in reply to knowing the sequence,

            I know all the adapter sequences used in the RNAseq reads from ILLUMINA. I have 27 of them, and know their sequences in full.

            To use trimmomatic I can use the ILLUMINACLIP:<adapters.fa>

            however, I do not know how to create the correct adapter.fa file in the correct format, only knowing the name.

            advice?

            Thank you

            Comment


            • #7
              You should make the adapter.fa in multi-fasta format.

              >Seq1
              ACTUAL_SEQUENCE
              >Seq2
              ACTUAL_SEQUENCE
              and so on.

              Remember to save it as text.

              Comment


              • #8
                Making the ILLUMINA adapter.fa file

                Hello.

                Thank you so very much for your response.


                The letter from illumina is given to the users as

                TruSeq Universal Adapter
                5’ AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
                TruSeq Adapter, Index 1 5
                5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG



                So based on what you are saying would I create the file as :

                >Universal Adapter 5’
                >AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
                >TruSeq Adapter, Index 1 5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG



                I am concerned about "whitespace" issues... because I am not sure if I should save the file as :

                TruSeq Universal Adapter
                >5’ AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
                TruSeq Adapter, Index 1
                >5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG



                my last question, is that ILLUMINACLIP requires adapter.fa.... but you suggest to save as .txt ?

                Could you post a clip of your adapter.fa so I can see how to format correctly?


                Thanks again

                (my other option is to update Trimmomatic from version .22 to version .32)

                Comment


                • #9
                  Multi fasta format has (2 or more) ID-sequence pairs. The ID line has to start with ">" and there should be no other ">" on that ID line. The sequence line has only sequence (no other characters).

                  Right format would be.

                  Code:
                  >TruSeq_Universal_Adapter
                  AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
                  >TruSeq_Adapter_Index 1
                  GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG
                  Though not strictly needed you can take out strange characters and spaces out of the sequence ID's.

                  I meant that you should save this file as plain text. You can use the name "adapter.fa" (if you include the quotes around the name in windows "save as") then no extension would be appended to that name but the file would still be in text format.

                  Comment


                  • #10
                    I see.

                    From the wiki, I was not sure if I needed to include all the barcode information.

                    thank you

                    Comment


                    • #11
                      Updated Trimmomatic

                      Originally posted by GenoMax View Post
                      You should make the adapter.fa in multi-fasta format.



                      and so on.

                      Remember to save it as text.
                      Hello. thank you for the response!

                      So I updated the Trimmomatic, and am not sure which adapter to use , which they provide for the users?

                      essentially, none of the adapters they provide, match with the list that I have been given from Illumina.

                      yet the adapters found in Trimmomatic don't seem to match any of the ones on the attached list.

                      NexteraPE
                      >PrefixNX/1
                      AGATGTGTATAAGAGACAG
                      >PrefixNX/2
                      AGATGTGTATAAGAGACAG
                      >Trans1
                      TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
                      >Trans1_rc
                      CTGTCTCTTATACACATCTGACGCTGCCGACGA
                      >Trans2
                      GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
                      >Trans2_rc
                      CTGTCTCTTATACACATCTCCGAGCCCACGAGAC




                      Essentially, the adapter from Truseq2- prefix/1 matches the universal adapter in the Illumina sheet.

                      However, I have multiple indices used from this sheet.

                      So how do I select the correct adapter from Trimmomatic?

                      Or how do I customize my own adapter sheet?
                      Attached Files

                      Comment


                      • #12
                        Originally posted by arcolombo698 View Post
                        Hello. thank you for the response!

                        So I updated the Trimmomatic, and am not sure which adapter to use , which they provide for the users?

                        essentially, none of the adapters they provide, match with the list that I have been given from Illumina.

                        yet the adapters found in Trimmomatic don't seem to match any of the ones on the attached list.

                        NexteraPE
                        >PrefixNX/1
                        AGATGTGTATAAGAGACAG
                        >PrefixNX/2
                        AGATGTGTATAAGAGACAG
                        >Trans1
                        TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
                        >Trans1_rc
                        CTGTCTCTTATACACATCTGACGCTGCCGACGA
                        >Trans2
                        GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
                        >Trans2_rc
                        CTGTCTCTTATACACATCTCCGAGCCCACGAGAC




                        Essentially, the adapter from Truseq2- prefix/1 matches the universal adapter in the Illumina sheet.

                        However, I have multiple indices used from this sheet.

                        So how do I select the correct adapter from Trimmomatic?

                        Or how do I customize my own adapter sheet?


                        Here is my custom adapter.fa. I will upload my FastQC report after i finish running it.

                        Here is my original fastqc report for a sample that HAS ADAPTERS in it, this is before i did the trimmomatic

                        [WARN] Overrepresented sequences
                        Sequence Count Percentage Possible Source
                        GCAGATAGTGAGGAAAGTTGAGCCAATAATGACGTGAAGTCCGTGGAAGC 52178 0.18044319834652642 No Hit
                        AGTAGTATAGTGATGCCAGCAGCTAGGACTGGGAGAGATAGGAGAAGTAG 51279 0.17733425520356333 No Hit
                        GCTTTGGCTCTCCTTGCAAAGTTATTTCTAGTTAATTCATTATGCAGAAG 49511 0.17122011562986064 No Hit
                        TTTGATGGTAAGGGAGGGATCGTTGACCTCGTCTGTTATGTAAAGGATGC 44215 0.15290536269867885 No Hit
                        GCCATATCGGGGGCACCGATTATTAGGGGAACTAGTCAGTTGCCAAAGCC 32007 0.11068736727121142 No Hit
                        GTCAGTTCAGTGTTTTAATCTGACGCAGGCTTATGCGGAGGAGAATGTTT 31149 0.10772021130162042 No Hit
                        TTGTCAGTTCAGTGTTTTAATCTGACGCAGGCTTATGCGGAGGAGAATGT 31053 0.10738822182250535 No Hit
                        AGCTTTGGCTCTCCTTGCAAAGTTATTTCTAGTTAATTCATTATGCAGAA 30693 0.10614326127582382 No Hit
                        AGTTAGATTTACGCCGATGAATATGATAGTGAAATGGATTTTGGCGTAGG 29276 0.10124295823513564 No Hit
                        TGGTCTAGGGTGTAGCCTGAGAATAGGGGAAATCAGTGAATGAAGCCTCC 29193 0.10095592566465071 No Hit

                        Comment


                        • #13
                          Custom Adapter.fa for Trimmomatic version .32

                          So here is my command to submit the Trimmomatic


                          java -classpath /auto/rcf-proj/sa1/software/Trimmomatic-0.32/trimmomatic-0.32.jar org.usadellab.trimmomatic.TrimmomaticPE -threads 16 -phred33 CHLA-15_S1_R1.fastq.gz CHLA-15_S1_R2.fastq.gz noadapter_paired_trimmed_CHLA-15_S1_R1.fastq.gz noadapter_unpaired_trimmed_CHLA-15_S1_R1.fastq.gz noadapter_paired_trimmed_CHLA-15_S1_R2.fastq.gz noadapter_unpaired_trimmed_CHLA-15_S1_R2.fastq.gz ILLUMINACLIP:/auto/rcf-proj/sa1/software/Trimmomatic-0.32/adapters/TrueSeq2-PE.fa:2:30:10 LEADING:3 TRAILING:3 HEADCROP:10 SLIDINGWINDOW:4:10 MINLEN:30



                          and here is my adapter file


                          >PrefixPE/1
                          AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
                          >PrefixPE/2
                          CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
                          >PCR_Primer1
                          AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
                          >PCR_Primer1_rc
                          AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT
                          >PCR_Primer2
                          CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
                          >PCR_Primer2_rc
                          AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG
                          >FlowCell1
                          TTTTTTTTTTAATGATACGGCGACCACCGAGATCTACAC
                          >FlowCell2
                          TTTTTTTTTTCAAGCAGAAGACGGCATACGA
                          >TruSeq_Adapter_Index1
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index2
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index3
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index4
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index5
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index6
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index7
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index8
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTGAATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index9
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index10
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index11
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index12
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index13
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTCAACAATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index14
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTTCCGTATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index15
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACATGTCAGAATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index16
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTCCCGATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index18
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCCGCACATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index19
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGAAACGATCTCGTATGCCGTCTTCTGCTTG
                          >TruSeq_Adapter_Index20
                          GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGGCCTTATCTCGTATGCCGTCTTCTGCTTG



                          awaiting results

                          Comment


                          • #14
                            So I am running the trimmomatic with my custom made adapter.fa file, and it should remove the over represented genes that FASTQC has shown.... update to arrive soon.

                            THank you in advance.

                            Comment


                            • #15
                              Trimmomatic is not cutting the adapters

                              Hello.

                              If you read the above commands, I submitted the trimmomatic commands, and it is not cutting the adapters.

                              Need some help here.

                              Comment

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