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  • You don't have your own data that you are trying to map?

    You need to provide/replace the "Sample*" names in the example command line with actual files you have. You can download some data from NCBI SRA if you don't have any of your own.

    Comment


    • Thank you very much for your kind reply. I have tried using the following ...

      Zillur-Rahman:saccharomyces ZILLURRAHMAN$ pwd
      /Users/ZILLURRAHMAN/desktop/phd/saccharomyces
      Zillur-Rahman:saccharomyces ZILLURRAHMAN$ ls
      ERR000004.fastq
      ERR000004_1.fastq
      ERR000004_2.fastq
      S.cervacie288c.fastq
      Saccharomyces_cerevisiae.R64-1-1.24.dna_rm.chromosome.Mito.fa
      Saccharomyces_cerevisiae_Ensembl_R64-1-1
      Saccharomyces_cerevisiae_Ensembl_R64-1-1.tar.gz
      chromosome.1.fa
      chromosome.2.fa
      chromosome.3.fa
      chromosome.4.fa
      chromosome.IX.fa
      chromosome.V.fa
      chromosome.VI.fa
      chromosome.VII.fa
      eg2.bam
      eg2.raw.bcf
      eg2.sam
      eg2.sorted.bam
      Zillur-Rahman:saccharomyces ZILLURRAHMAN$ bowtie2 -x saccharomyces_cerevisiae_ensembl_r64-1-1/saccharomyces_cerevisiae/ensembl/r64-1-1/sequence/bowtie2index/genome -1 err000004_1.fastq -2 err000004_2.fastq -S eg2.sam

      Zillur-Rahman:saccharomyces ZILLURRAHMAN$ samtools view -bS eg2.sam > eg2.bam


      Zillur-Rahman:saccharomyces ZILLURRAHMAN$ samtools sort eg2.bam eg2.sorted


      Zillur-Rahman:saccharomyces ZILLURRAHMAN$ samtools mpileup -uf saccharomyces_cerevisiae_ensembl_r64-1-1/saccharomyces_cerevisiae/ensembl/r64-1-1/sequence/bowtie2index/genome.fa eg2.sorted.bam | bcftools view -bvcg - > eg2.raw.bcf



      Zillur-Rahman:saccharomyces ZILLURRAHMAN$ head eg2.raw.bcf
      Zillur-Rahman:saccharomyces ZILLURRAHMAN$ bcftools stats eg2.raw.bcf
      [vcf.c:1224 vcf_hdr_read] Could not read the header
      Could not read the file or the file is not indexed: eg2.raw.bcf
      Zillur-Rahman:saccharomyces ZILLURRAHMAN$


      My sam, bam, sorted bam everything is running, but eg2.raw.bcf is not working. May be I am missing some point....

      Best Regards
      Zillur

      Comment


      • You need to convert the bcf file to a text format vcf file. Following is just an example.

        Code:
        $ bcftools view var.raw.bcf | vcfutils.pl varFilter -D100 > var.flt.vcf

        Comment


        • Thank you very much for your kind reply. I have tried but....
          Is it the problem of my eg2.raw.bcf? After this command "samtools mpileup -uf ...." it is showing that "fail to open FASTA index."

          Best Regards
          Zillur


          Zillur-Rahman:saccharomyces ZILLURRAHMAN$ samtools mpileup -uf ../saccharomyces_cerevisiae_ensembl_r64-1-1/saccharomyces_cerevisiae/ensembl/r64-1-1/sequence/bowtie2index/genome eg2.sorted.bam | bcftools view -bvcg - > eg2.raw.bcf
          bcftools: invalid option -- b

          About: VCF/BCF conversion, view, subset and filter VCF/BCF files.
          Usage: bcftools view [options] <in.vcf.gz> [region1 [...]]

          Output options:
          -G, --drop-genotypes drop individual genotype information (after subsetting if -s option set)
          -h/H, --header-only/--no-header print the header only/suppress the header in VCF output
          -l, --compression-level [0-9] compression level: 0 uncompressed, 1 best speed, 9 best compression [-1]
          -o, --output-file <file> output file name [stdout]
          -O, --output-type <b|u|z|v> b: compressed BCF, u: uncompressed BCF, z: compressed VCF, v: uncompressed VCF [v]
          -r, --regions <region> restrict to comma-separated list of regions
          -R, --regions-file <file> restrict to regions listed in a file
          -t, --targets [^]<region> similar to -r but streams rather than index-jumps. Exclude regions with "^" prefix
          -T, --targets-file [^]<file> similar to -R but streams rather than index-jumps. Exclude regions with "^" prefix

          Subset options:
          -a, --trim-alt-alleles trim alternate alleles not seen in the subset
          -I, --no-update do not (re)calculate INFO fields for the subset (currently INFO/AC and INFO/AN)
          -s, --samples [^]<list> comma separated list of samples to include (or exclude with "^" prefix)
          -S, --samples-file [^]<file> file of samples to include (or exclude with "^" prefix)
          --force-samples only warn about unknown subset samples

          Filter options:
          -c/C, --min-ac/--max-ac <int>[:<type>] minimum/maximum count for non-reference (nref), 1st alternate (alt1) or minor (minor) alleles [nref]
          -f, --apply-filters <list> require at least one of the listed FILTER strings (e.g. "PASS,.")
          -g, --genotype [^]<hom|het|miss> require one or more hom/het/missing genotype or, if prefixed with "^", exclude sites with hom/het/missing genotypes
          -i/e, --include/--exclude <expr> select/exclude sites for which the expression is true (see man page for details)
          -k/n, --known/--novel select known/novel sites only (ID is not/is '.')
          -m/M, --min-alleles/--max-alleles <int> minimum/maximum number of alleles listed in REF and ALT (e.g. -m2 -M2 for biallelic sites)
          -p/P, --phased/--exclude-phased select/exclude sites where all samples are phased
          -q/Q, --min-af/--max-af <float>[:<type>] minimum/maximum frequency for non-reference (nref), 1st alternate (alt1) or minor (minor) alleles [nref]
          -u/U, --uncalled/--exclude-uncalled select/exclude sites without a called genotype
          -v/V, --types/--exclude-types <list> select/exclude comma-separated list of variant types: snps,indels,mnps,other [null]
          -x/X, --private/--exclude-private select/exclude sites where the non-reference alleles are exclusive (private) to the subset samples

          [fai_load] build FASTA index.
          [fai_build] fail to open the FASTA file ../saccharomyces_cerevisiae_ensembl_r64-1-1/saccharomyces_cerevisiae/ensembl/r64-1-1/sequence/bowtie2index/genome
          [fai_load] fail to open FASTA index.
          Zillur-Rahman:saccharomyces ZILLURRAHMAN$ bcftools view eg2.raw.bcf | vcfutils.pl varFilter -D100 > eg2.flt.vcf
          [vcf.c:1224 vcf_hdr_read] Could not read the header
          Failed to open or the file not indexed: eg2.raw.bcf
          Zillur-Rahman:saccharomyces ZILLURRAHMAN$ ls
          ERR000004.fastq
          ERR000004_1.fastq
          ERR000004_2.fastq
          S.cervacie288c.fastq
          Saccharomyces_cerevisiae.R64-1-1.24.dna_rm.chromosome.Mito.fa
          Saccharomyces_cerevisiae_Ensembl_R64-1-1
          Saccharomyces_cerevisiae_Ensembl_R64-1-1.tar.gz
          chromosome.1.fa
          chromosome.2.fa
          chromosome.3.fa
          chromosome.4.fa
          chromosome.IX.fa
          chromosome.V.fa
          chromosome.VI.fa
          chromosome.VII.fa
          eg2.bam
          eg2.flt.vcf
          eg2.raw.bcf
          eg2.sam
          eg2.sorted.bam
          var.flt.vcf
          Zillur-Rahman:saccharomyces ZILLURRAHMAN$

          Comment


          • Thank you very much for your kind reply and I am sorry to disturb you repeatedly. I have done this in a several way. But there is problem in my file....

            Zillur-Rahman:saccharomyces ZILLURRAHMAN$ bcftools stats eg2.flt.vcf
            Zillur-Rahman:saccharomyces ZILLURRAHMAN$ bcftools stats eg2.raw.bcf
            Zillur-Rahman:saccharomyces ZILLURRAHMAN$ bcftools stats u

            All give me the same result:

            # This file was produced by bcftools stats (1.1+htslib-1.1) and can be plotted using plot-vcfstats.
            # The command line was: bcftools stats u
            #
            # Definition of sets:
            # ID [2]id [3]tab-separated file names
            ID 0 u
            # SN, Summary numbers:
            # SN [2]id [3]key [4]value
            SN 0 number of samples: 1
            SN 0 number of records: 0
            SN 0 number of SNPs: 0
            SN 0 number of MNPs: 0
            SN 0 number of indels: 0
            SN 0 number of others: 0
            SN 0 number of multiallelic sites: 0
            SN 0 number of multiallelic SNP sites: 0
            # TSTV, transitions/transversions:
            # TSTV [2]id [3]ts [4]tv [5]ts/tv [6]ts (1st ALT) [7]tv (1st ALT) [8]ts/tv (1st ALT)
            TSTV 0 0 0 0.00 0 0 0.00
            # Sis, Singleton stats:
            # SiS [2]id [3]allele count [4]number of SNPs [5]number of transitions [6]number of transversions [7]number of indels [8]repeat-consistent [9]repeat-inconsistent [10]not applicable
            SiS 0 1 0 0 0 0 0 0 0
            # AF, Stats by non-reference allele frequency:
            # AF [2]id [3]allele frequency [4]number of SNPs [5]number of transitions [6]number of transversions [7]number of indels [8]repeat-consistent [9]repeat-inconsistent [10]not applicable
            # QUAL, Stats by quality:
            # QUAL [2]id [3]Quality [4]number of SNPs [5]number of transitions (1st ALT) [6]number of transversions (1st ALT) [7]number of indels
            # IDD, InDel distribution:
            # IDD [2]id [3]length (deletions negative) [4]count
            # ST, Substitution types:
            # ST [2]id [3]type [4]count
            ST 0 A>C 0
            ST 0 A>G 0
            ST 0 A>T 0
            ST 0 C>A 0
            ST 0 C>G 0
            ST 0 C>T 0
            ST 0 G>A 0
            ST 0 G>C 0
            ST 0 G>T 0
            ST 0 T>A 0
            ST 0 T>C 0
            ST 0 T>G 0

            Here is my files:

            Zillur-Rahman:saccharomyces ZILLURRAHMAN$ ls
            ERR000004.fastq
            ERR000004_1.fastq
            ERR000004_2.fastq
            IV.fa
            IX.fa
            MT.fa
            S.cervacie288c.fastq
            Saccharomyces_cerevisiae.R64-1-1.24.dna_rm.chromosome.Mito.fa
            Saccharomyces_cerevisiae_Ensembl_R64-1-1
            Saccharomyces_cerevisiae_Ensembl_R64-1-1.tar.gz
            V.fa
            VI.fa
            VII.fa
            VIII.fa
            X.fa
            XI.fa
            XII.fa
            XIII.fa
            XIV.fa
            XV.fa
            XVI.fa
            ch_1.fa
            ch_2.fa
            ch_3.fa
            chromosome.1.fa
            chromosome.2.fa
            chromosome.3.fa
            chromosome.4.fa
            chromosome.IX.fa
            chromosome.V.fa
            chromosome.VI.fa
            chromosome.VII.fa
            eg2.bam
            eg2.flt.vcf
            eg2.raw.bcf
            eg2.sam
            eg2.sorted.bam
            genome.1.bt2
            genome.2.bt2
            genome.3.bt2
            genome.4.bt2
            genome.fa
            genome.fa.fai
            genome.rev.1.bt2
            genome.rev.2.bt2
            u
            Zillur-Rahman:saccharomyces ZILLURRAHMAN$
            I have got the final output by:

            Zillur-Rahman:saccharomyces ZILLURRAHMAN$ bcftools view eg2.raw.bcf | vcfutils.pl varFilter -D100 > eg2.flt.vcf
            Zillur-Rahman:saccharomyces ZILLURRAHMAN$ bcftools view u | vcfutils.pl varFilter -D100 > eg2.flt.vcf

            But all gave me the same result. Is there any problem with my downloaded file?

            Best Regards
            Zillur

            Comment


            • Looks like you made the bcf file using the new version of bcftools (v.1.1). The options for that have changed (example I had included for older version of bcftools).

              Have you checked that the alignment file you have looks ok (with something like IGV)?

              Comment


              • Thank you very much for your kind reply. Here my sam and bam files......
                Does it seem ok?

                Best Regards
                Zillur


                9?Rp?r???D"??0~??3j?BCZillur-Rahman:saccharomyces ZILLURRAHMAN$ head eg2.sam
                @HD VN:1.0 SO:unsorted
                @SQ SN:I LN:230218
                @SQ SN:II LN:813184
                @SQ SN:III LN:316620
                @SQ SN:IV LN:1531933
                @SQ SN:IX LN:439888
                @SQ SN:MT LN:85779
                @SQ SN:V LN:576874
                @SQ SN:VI LN:270161
                @SQ SN:VII LN:1090940
                Zillur-Rahman:saccharomyces ZILLURRAHMAN$ head eg2.bam
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                9?Rp?r???D"??0~??3j?BCZillur-Rahman:saccharomyces ZILLURRAHMAN$

                Comment


                • If you were able to paste the contents of your bam file (which is binary so no human readable text there) in the post above then something is not right.

                  Can you post output of command below?

                  Code:
                  $ ls -lh *.fastq eg2.*

                  Comment


                  • Thank you very much. Here it is...

                    Zillur-Rahman:saccharomyces ZILLURRAHMAN$ ls -lh *.fastq eg2.*
                    -rw-r--r--@ 1 ZILLURRAHMAN staff 493M Nov 14 11:45 ERR000004.fastq
                    -rw-r--r--@ 1 ZILLURRAHMAN staff 60K Nov 14 11:45 ERR000004_1.fastq
                    -rw-r--r--@ 1 ZILLURRAHMAN staff 60K Nov 14 11:46 ERR000004_2.fastq
                    -rw-r--r--@ 1 ZILLURRAHMAN staff 498M Nov 12 15:09 S.cervacie288c.fastq
                    -rw-r--r-- 1 ZILLURRAHMAN staff 35K Dec 11 01:00 eg2.bam
                    -rw-r--r-- 1 ZILLURRAHMAN staff 2.3K Dec 11 01:28 eg2.flt.vcf
                    -rw-r--r-- 1 ZILLURRAHMAN staff 2.3K Dec 11 01:08 eg2.raw.bcf
                    -rw-r--r-- 1 ZILLURRAHMAN staff 187K Dec 11 00:59 eg2.sam
                    -rw-r--r-- 1 ZILLURRAHMAN staff 35K Dec 11 09:39 eg2.sam.bam
                    -rw-r--r-- 1 ZILLURRAHMAN staff 36K Dec 11 09:39 eg2.sam.bam_sorted.bam
                    -rw-r--r-- 1 ZILLURRAHMAN staff 15K Dec 11 09:39 eg2.sam.bam_sorted.bam.bai
                    -rw-r--r-- 1 ZILLURRAHMAN staff 44M Dec 11 09:39 eg2.sam.ugenedb
                    -rw-r--r-- 1 ZILLURRAHMAN staff 36K Dec 11 01:01 eg2.sorted.bam

                    Comment


                    • How did you derive ERR000004_1.fastq and ERR000004_2.fastq? Did they come from this file ERR000004.fastq? Why are they so small compared to ERR000004.fastq?

                      Comment


                      • Thank you very much. I have downloaded these from http://www.ebi.ac.uk/about/news/serv...na-release-122

                        Yes, I think err000004_1.fastq and err000004_2.fastq came from err000004.fastq but why they are small compared to err000004.fastq, I don't know.

                        Best Regards
                        Zillur

                        Comment


                        • I can't easily determine what those three files mean from the ENA record. What exactly are you trying to do?

                          Comment


                          • Thank you very much for your kind reply. I have to map yeast genome downloading a good sequence from authentic source to find SNP using bowtie2. Can you give me some suggestions. I am very much sorry that I am doing this mess.

                            Best Regards
                            Zillur

                            Comment


                            • If you don't have a specific aim then go back to your dataset. Use only the large file as single end dataset. Repeat the analysis.

                              Comment


                              • Thank you very much for your kind reply and suggestions. Can you give me some specific link to download yeast genome to analyse by bowtie2?

                                Ensembl Fungi is a genome-centric portal for fungal species of scientific interest


                                The Saccharomyces Genome Database (SGD) provides comprehensive integrated biological information for the budding yeast Saccharomyces cerevisiae.


                                I have seen these but can't working with them. May be I am downloading the wrong data...

                                Best Regards
                                Zillur

                                Comment

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