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Unmapped_Reads_In_a_Samfile.md

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Unmapped Reads in a Samfile

Please take note, you should have a BEOCAT account or samtools downloaded for this exercise.

Part 1:

Part 2:

Filter SAM files to create file with only unmapped reads using the Samtools flag filter.

/homes/bioinfo/bioinfo_software/samtools-0.1.18/samtools view -S -f4 /homes/bioinfo/test_de_novo_DE/BT20_rep1_200.sam > ~/unmapped/BT20_rep1_200.unmapped.sam

Read more on flag here. Read more on samtools here.

Part 3:

Convert SAM to fastq

java -jar ~/bioinfo_software/picard-tools-1.112/SamToFastq.jar INPUT=/homes/bioinfo/unmapped/BT20_rep1_200.unmapped.sam FASTQ=/homes/bioinfo/unmapped/BT20_rep1_200.unmapped_1.fastq SECOND_END_FASTQ=/homes/bioinfo/unmapped/BT20_rep1_200.unmapped_2.fastq

Read more on picard here.

Note:

Picard-tools-1.112 does not like relative paths so use the absolute path in file names.

e.g.

/homes/jfellers/unmapped/filename

rather than

"~/unmapped/filename"

Part 3:

Check for broken pairs This simple script, broken_pair_removal.pl, finds pairs that have been broken and moves them to a separate file as single end reads. The script takes the full path of your forward and reverse reads followed by full path of your forward and reverse read output files.

perl /homes/sheltonj/abjc/scripts/broken_pair_removal.pl /homes/bioinfo/unmapped/BT20_rep1_200.unmapped_1.fastq /homes/bioinfo/unmapped/BT20_rep1_200.unmapped_2.fastq /homes/bioinfo/unmapped/BT20_rep1_200.unmapped_2_bp.fastq /homes/bioinfo/unmapped/BT20_rep1_200.unmapped_2_bp.fastq