Truncated bamtofastq output files.

Question

[mirza]

Hi, I am working with a mixed RNASeq data of plant and fungus. I aligned my Illumina paired-end reads using bowtie2, to one of the reference. I got mapped and unmapped.bam files. Now, I want to map these unmapped reads back to the second organism reference. I used samtools to sort the files by name using

$ samtools sort -n my_file_unmapped.bam -o my_file_unmapped.qsort -O BAM

and got, unmapped.qsort files. Visualized the files using samtools view,

$  samtools view Rs_Os1_1d_unmapped.qsort

D6B775P1:106:C4NARACXX:7:1101:2383:96122    69  *   0   255 *   *   0   0   CGGTGTTAGGCTTTCCATGACGATCGGTGTGGGAGGTGCCGTAGTTGTCAGTTGTGGACCCGGCACCGTAGCCGGCGGTAGTACTGCTGGTACCAGTAGT    BBBFFFFFFFFFFIIIIIIIIIIIIIIFFFFIIIIIFFIIIBFFIIIIIIIIIIIFFFFFFFFBFFFFFFFFFFFBFFBBFFFFFFFFFFFFFFFFFFFB
D6B775P1:106:C4NARACXX:7:1101:2383:96122    133 *   0   255 *   *   0   0   CAATCACAGGACGACAAATACCTACGGGTCGTCGAACACCAGTGACAACTACGGTTCAACTGACCGCACTCGCGGCACTGATCTGAACACCTGAACCGGA    BBBFFFFFFFFFFIIIIIIIIIIIIIIIBFFIIIIIFIIIIIFFFFFFFFBFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
D6B775P1:106:C4NARACXX:7:1101:2384:9664 69  *   0   255 *   *   0   0   AACACTGCTCGAGGTGCCATCGCGGATCGTCAGGCTATTGCCGAAGCTCTAAAGAGCGGGCAGATCAATGGATACGCCGGAGACGTGTGGGACGTTCAGC    BBBFFFFFFFFFFIFIIIIIIIIIIIIIIFIIIIIIIIIIIIFIFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFBFFFFFFFFBBFBFFFFFF<BFFFF
D6B775P1:106:C4NARACXX:7:1101:2384:9664 133 *   0   255 *   *   0   0   CCAGAGTAGTGGGGAACCATTCCGTTTCCTCCACCAAGAGGGTTCTTCATCGTGCGCCATGGATGGTCAGCAGGCGCTGGCTGAACGTCCCACACGTCTC    BBBFFFFFFFFFFIIIIIIIIIIIIIIIIIIIIIIIIIIIIIFIIIIIIIFFIIIIIIFFFFFFFFBFFFFFFFFFFFFFFFFFFBFBFFFFFFFFFFFF

Then I tried converting the .bam files to .fastq using bedtools bedtofastq

$ bedtools bamtofastq -i my_file_unmapped.qsort -fq my_file_unmapped.end1.fq -fq2 my_file_unmapped.end2.fq

Got 2 output files, my_file_unmapped.end1.fq my_file_unmapped.end2.fq But when I tried to visualize the file using samtools view, using the command

$ samtools view my_files_unmapped.end1.fq

I am getting the following error, [W::sam_read1] parse error at line 2; [main_samview] truncated file.

What am I doing wrong??

[GenoMax]

Why are you using samtools to view a plain fastq file (if the files have indeed been generated correctly)?

[Prasad]

samtool view is not meant for fastq files. it is used for alignment files

[mirza]

Oh, now this is indeed a silly mistake. I should simply use "more" to visualize a fastq file in terminal.

[Prasad]

use less filename command

[mirza]

@Prasad ok, thanks Prasad.

Answer 1

[GenoMax]

Use BBSplit from BBMap as described here: BBSplit syntax for generating builds for the reference genome and how to call different builds.

[GiV17]

yes, but after I use BBsplit, I obtain the fastq file and then I can to remap this with STAR but the count? FeatureCount doesn't work well with bbsplit. So my question is: After BBsplit What can I use to map and to calculate the count? Thanks

Answer 2

[agata88]

Why don't you just map again whole fastq files to other reference? First mapping to fungi and second to plant. Without parsing the unmapped bam files.

[mirza]

In such mixed data, sometimes reads are wrongly mapped to a reference and we get to know only when we go for annotation. I think i'll be better off using this approach.