[GenoMax]

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?

[zillur]

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

[GenoMax]

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?

$ ls -lh *.fastq eg2.*

[zillur]

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
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9?Rp?r???D"??0~??3j?BCZillur-Rahman:saccharomyces ZILLURRAHMAN$

[GenoMax]

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)?

[zillur]

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

[zillur]

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$

[GenoMax]

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

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

[zillur]

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

[GenoMax]

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.

[zillur]

Thank you very much for your kind reply. But where I get the sample fast files?

Best Regards
Zillur

Zillur-Rahman:bowtie2index ZILLURRAHMAN$ ls
eg2.sam genome.3.bt2 genome.fa.fai
genome.1.bt2 genome.4.bt2 genome.rev.1.bt2
genome.2.bt2 genome.fa genome.rev.2.bt2
Zillur-Rahman:bowtie2index ZILLURRAHMAN$ $ bowtie2 -x genome -1 Sample_R1.fastq -2 Sample_R2.fastq -S eg2.sam
-bash: $: command not found
Zillur-Rahman:bowtie2index ZILLURRAHMAN$ bowtie2 -x genome -1 Sample_R1.fastq -2 Sample_R2.fastq -S eg2.sam
Warning: Could not open read file "Sample_R1.fastq" for reading; skipping...
Error: No input read files were valid
(ERR): bowtie2-align exited with value 1
Zillur-Rahman:bowtie2index ZILLURRAHMAN$ bowtie2 -x genome -1 ../Sample_R1.fastq -2 ../Sample_R2.fastq -S eg2.sam
Warning: Could not open read file "../Sample_R1.fastq" for reading; skipping...
Error: No input read files were valid
(ERR): bowtie2-align exited with value 1
Zillur-Rahman:bowtie2index ZILLURRAHMAN$ pwd
/Users/ZILLURRAHMAN/desktop/phd/saccharomyces/saccharomyces_cerevisiae_ensembl_r64-1-1/saccharomyces_cerevisiae/ensembl/r64-1-1/sequence/bowtie2index
Zillur-Rahman:bowtie2index ZILLURRAHMAN$

[GenoMax]

You need to use fastq format files (which would be your sample) against the yeast genome to do the mapping. In the command line you had posted above you are using yeast chromosome files (which does not make any sense). Replace them with your sample fastq files.

"genome" ==> Entire yeast genome/all chromosomes together.

If you have paired end data:

$ bowtie2 -x /path_to_bowtie2index_dir/genome -1 Sample_R1.fastq -2 Sample_R2.fastq -S eg2.sam

[zillur]

Thank you very much for your kind reply. I have tried this...

Zillur-Rahman:sequence ZILLURRAHMAN$ ls
AbundantSequences Bowtie2Index Chromosomes
BWAIndex BowtieIndex WholeGenomeFasta
Zillur-Rahman:sequence ZILLURRAHMAN$ cd bowtie2index
Zillur-Rahman:bowtie2index ZILLURRAHMAN$ ls
eg2.sam genome.3.bt2 genome.fa.fai
genome.1.bt2 genome.4.bt2 genome.rev.1.bt2
genome.2.bt2 genome.fa genome.rev.2.bt2
Zillur-Rahman:bowtie2index ZILLURRAHMAN$ bowtie2 -x genome -1 ../chromosomes/ch_1.fa -2 ../chromosomes/ch_2.fa -S eg2.sam
Error: reads file does not look like a FASTQ file
libc++abi.dylib: terminating with uncaught exception of type int
(ERR): bowtie2-align died with signal 6 (ABRT)
Zillur-Rahman:bowtie2index ZILLURRAHMAN$


What should I do if I want to map all chromosomes together? There are 16 chromosomes

Best Regards
Zillur

[GenoMax]

Your command can be as follows:

$ bowtie2 -x /path_to_bowtie2index_dir/genome -1 Sample_R1.fastq -2 Sample_R2.fastq -S eg2.sam

[GenoMax]

If you downloaded the iGenomes files the Bowtie2index will have a generic "base" name. It is going to be "genome". This is the prefix (part before the first period in file name) from the index files.