<p>I think cuffdiff is not quite giving you the right error...it looks like the crux of your problem is you aligned each sample to a different reference.  </p>

CC BY-SA 4.0
2019-06-07T20:39:19.560Z
2019-06-07T20:39:19.560Z
swbarnes2
<p>As @swbarnes2 noted, the error message is because the chromosome names, sizes, and orders differ between the two reference genomes/species you aligned against. Even if they were the same, you're comparing one sample against another between organisms, which is innately not something likely to work well. Further, I wouldn't expect the Zea Mays annotation to be very useful with whatever <code>Ref_Lai</code> is.</p>

CC BY-SA 4.0
2019-06-08T08:19:07.777Z
2019-06-08T08:19:07.777Z
Devon Ryan
<p>I am a novice to bioinformatics trying to run a cuffdiff job in cufflinks and keep getting this error message: </p>

<pre><code>Error: sort order of reads in BAMs must be the same
</code></pre>

<p><strong>GENERATING SAM FILES:</strong></p>

<pre><code>cd /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_hisat2_11-5
module load hisat2  
hisat2 -k 1 -p 8 -S 11-5_B73_accepted_hits.sam -x /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_Zea_mays.AGPv4.dna.toplevel -1 /fs/project/PMIU0157/Claire_Stoll/RNASeq/11-5_FCHHL7JBBXX_L7_DLPRMzP300H3AAAPEI-27_1.fq  -2 /fs/project/PMIU0157/Claire_Stoll/RNASeq/11-5_FCHHL7JBBXX_L7_DLPRMzP300H3AAAPEI-27_2.fq


cd /fs/project/PMIU0157/Claire_Stoll/RNASeq/Mo17_hisat2/Mo17_hisat2_11R-5
module load hisat2  
hisat2 -k 1 -p 8 -S 11-5_Mo17_accepted_hits.sam -x /fs/project/PMIU0157/Claire_Stoll/RNASeq/Mo17_hisat2//Mol17_Ref_Lai -1 /fs/project/PMIU0157/Claire_Stoll/RNASeq/11R-5_DLPRMzP300B4AAAPEI-2_1.fq  -2 /fs/project/PMIU0157/Claire_Stoll/RNASeq/11R-5_DLPRMzP300B4AAAPEI-2_2.fq
</code></pre>

<p><strong>CONVERTING SAM TO BAM:</strong> </p>

<pre><code>cd /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_hisat2_11-5
module load samtools 
samtools view -bS a_B73_11-5_accepted_hits.sam &gt; a_B73_11-5_accepted_hits.bam
samtools sort a_B73_11-5_accepted_hits.bam -o a_B73_11-5_accepted_hits.sorted.bam

cd /fs/project/PMIU0157/Claire_Stoll/RNASeq/Mo17_hisat2/Mo17_hisat2_11-5
module load samtools 
samtools view -bS a_Mo17_11-5_accepted_hits.sam &gt; a_Mo17_11-5_accepted_hits.bam
samtools sort a_Mo17_11-5_accepted_hits.bam -o a_Mo17_11-5_accepted_hits.sorted.bam
</code></pre>

<p><strong>CUFFDIFF JOB</strong></p>

<pre><code>cd /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_hisat2_11-5
module load cufflinks/2.2.1
cuffdiff -b /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_hisat2_11-5 -u -N -p 8 /fs/project/PMIU0157/Claire_Stoll/Zea_mays.AGPv4.36.chr.gff3 /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_hisat2_11-5/a_B73_11-5_accepted_hits.sorted.bam /fs/project/PMIU0157/Claire_Stoll/RNASeq/Mo17_hisat2/Mo17_hisat2_11-5/a_Mo17_11-5_accepted_hits.sorted.bam
</code></pre>

CC BY-SA 4.0
2019-06-07T19:04:27.863Z
Is it the last command that gives the error?
terdon
<phylogenetics><sam>
Error: sort order of reads in BAMs must be the same
# Error: sort order of reads in BAMs must be the same

(Asked by: username_0 on 2019-06-07)

I am a novice to bioinformatics trying to run a cuffdiff job in cufflinks and keep getting this error message: 
Error: sort order of reads in BAMs must be the same

GENERATING SAM FILES:
cd /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_hisat2_11-5
module load hisat2  
hisat2 -k 1 -p 8 -S 11-5_B73_accepted_hits.sam -x /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_Zea_mays.AGPv4.dna.toplevel -1 /fs/project/PMIU0157/Claire_Stoll/RNASeq/11-5_FCHHL7JBBXX_L7_DLPRMzP300H3AAAPEI-27_1.fq  -2 /fs/project/PMIU0157/Claire_Stoll/RNASeq/11-5_FCHHL7JBBXX_L7_DLPRMzP300H3AAAPEI-27_2.fq


cd /fs/project/PMIU0157/Claire_Stoll/RNASeq/Mo17_hisat2/Mo17_hisat2_11R-5
module load hisat2  
hisat2 -k 1 -p 8 -S 11-5_Mo17_accepted_hits.sam -x /fs/project/PMIU0157/Claire_Stoll/RNASeq/Mo17_hisat2//Mol17_Ref_Lai -1 /fs/project/PMIU0157/Claire_Stoll/RNASeq/11R-5_DLPRMzP300B4AAAPEI-2_1.fq  -2 /fs/project/PMIU0157/Claire_Stoll/RNASeq/11R-5_DLPRMzP300B4AAAPEI-2_2.fq

CONVERTING SAM TO BAM: 
cd /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_hisat2_11-5
module load samtools 
samtools view -bS a_B73_11-5_accepted_hits.sam > a_B73_11-5_accepted_hits.bam
samtools sort a_B73_11-5_accepted_hits.bam -o a_B73_11-5_accepted_hits.sorted.bam

cd /fs/project/PMIU0157/Claire_Stoll/RNASeq/Mo17_hisat2/Mo17_hisat2_11-5
module load samtools 
samtools view -bS a_Mo17_11-5_accepted_hits.sam > a_Mo17_11-5_accepted_hits.bam
samtools sort a_Mo17_11-5_accepted_hits.bam -o a_Mo17_11-5_accepted_hits.sorted.bam

CUFFDIFF JOB
cd /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_hisat2_11-5
module load cufflinks/2.2.1
cuffdiff -b /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_hisat2_11-5 -u -N -p 8 /fs/project/PMIU0157/Claire_Stoll/Zea_mays.AGPv4.36.chr.gff3 /fs/project/PMIU0157/Claire_Stoll/RNASeq/B73_hisat2/B73_hisat2_11-5/a_B73_11-5_accepted_hits.sorted.bam /fs/project/PMIU0157/Claire_Stoll/RNASeq/Mo17_hisat2/Mo17_hisat2_11-5/a_Mo17_11-5_accepted_hits.sorted.bam

**Tags:** phylogenetics, sam
> **Comments:**
> **username_1**: Is it the last command that gives the error?  

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## 2 answers

### Answer by username_2 on 2019-06-07. Score: 3

I think cuffdiff is not quite giving you the right error...it looks like the crux of your problem is you aligned each sample to a different reference.


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### Answer by username_3 on 2019-06-08. Score: 2

As @swbarnes2 noted, the error message is because the chromosome names, sizes, and orders differ between the two reference genomes/species you aligned against. Even if they were the same, you're comparing one sample against another between organisms, which is innately not something likely to work well. Further, I wouldn't expect the Zea Mays annotation to be very useful with whatever `Ref_Lai` is.


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