**340**wrote:

Dear all,

I want to perform differential expression analysis between two mirSeq samples.

This is not my own project. I received bam files in the following format. However it is more similar to count file than alignment file:

```
QNAME FLAG RNAME POS MAPQ CIGAR MRNM MPOS ISIZE SEQ QUAL OPT
@HD VN:1.0 SO:coordinate
@SQ SN:hsa-let-7a-1 LN:80
@SQ SN:hsa-let-7a-2 LN:72
@SQ SN:hsa-let-7a-3 LN:74
@SQ SN:hsa-let-7b LN:83
@SQ SN:hsa-let-7c LN:84
@SQ SN:hsa-let-7d LN:87
@SQ SN:hsa-let-7e LN:79
@SQ SN:hsa-let-7f-1 LN:87
@SQ SN:hsa-let-7f-2 LN:83
@SQ SN:hsa-mir-15a LN:83
@SQ SN:hsa-mir-16-1 LN:89
@SQ SN:hsa-mir-17 LN:84
@SQ SN:hsa-mir-18a LN:71
@SQ SN:hsa-mir-19a LN:82
@SQ SN:hsa-mir-19b-1 LN:87
@SQ SN:hsa-mir-19b-2 LN:96
@SQ SN:hsa-mir-20a LN:71
@SQ SN:hsa-mir-21 LN:72
@SQ SN:hsa-mir-22 LN:85
@SQ SN:hsa-mir-23a LN:73
```

Can you tell me the best way to get differential expressed miRNAs from these files?

Deseq2 does not recognize these files as count files.

I am looking for your comments

Nazanin

**0**• written 12 days ago by nazaninhoseinkhan •

**340**

Is this indeed a BAM file? It looks like it but should not contain a header explaining the columns. What is the output of

`samtools view your.bam | head`

and`samtools view -H your.bam | head`

9.3kCan you elaborate how you quantified the aligned file and what tools have been used? This does not look like a count matrix.

RNAseq pipeline using feature counts and DESeq2.

650It doesn't matter which aligner, its looks like a BAM file. Can you do featurecounts with your Bam file and extract the read counts. One more thing, if you want to use DESEQ2 for DE you have to have a replicates, but you said you have two samples, so better to go with Noiseq.

130