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rna paired sequence sort is important

i have rna paired fasta file I trimmed it using trim galore and then mapped it with hg38 bowtie2 index now i have sam file is it important to sort it for htseq count i have to ask that the process that i follow till now is it correct differential expression and second that what next to do to get differential expression i m new please help me

rna-seq sequence alignment
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updated by WouterDeCoster WouterDeCoster • written by sarfrajiiet • sarfrajiiet

Please add some punctuation and write in separate sentences. This is barely readable.

mapped it with hg38 bowtie2 index

bowtie2 does not due spliced alignment, but you did not tell us which organism you are working on. For eukaryotic organisms, STAR or HISAT2 would be a better choice.

is it important to sort it for htseq count

Yes. A better tool would be featureCounts.

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written by WouterDeCoster WouterDeCoster

1 answer

is it important to sort it for htseq count

Yes you should (always) sort your bam files.

Note that you can often do this directly after alignment without creating intermediate files:

aligner genome.fasta reads1.fasta.gz reads2.fastq.gz | samtools sort -o sorted_alignment.bam

A better tool for counting expression levels would be featureCounts.

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written by WouterDeCoster WouterDeCoster

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