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STAR number of input reads | 0

Hi, I am just learning RNA-seq analysis, I am trying to use STAR (2.7.11b) to align Drosophila fastq files. I have confirmed that my paths, permissions, index, and fastq files are correct, but the log_final.out shows that no reads were read.here is part of logs:

                      Number of input reads |   0
                  Average input read length |   0  
       and:
          --sjdbOverhang = 149 taken from the generated genome
        Started loading the genome: Wed Jul 22 12:07:03 2026

        Genome: size given as a parameter = 644878196
          SA: size given as a parameter = 1324986171
 SAindex: size given as a parameter = 1
Read from SAindex: pGe.gSAindexNbases=10  nSAi=1398100
nGenome=644878196;  nSAbyte=1324986171
GstrandBit=32   SA number of indices=321208768
Shared memory is not used for genomes. Allocated a private copy of the genome.
Genome file size: 644878196 bytes; state: good=1 eof=0 fail=0 bad=0
Loading Genome ... done! state: good=1 eof=0 fail=0 bad=0; loaded 644878196 bytes
SA file size: 1324986171 bytes; state: good=1 eof=0 fail=0 bad=0
Loading SA ... done! state: good=1 eof=0 fail=0 bad=0; loaded 1324986171 bytes
Loading SAindex ... done: 6116787 bytes
Finished loading the genome: Wed Jul 22 12:07:05 2026

Processing splice junctions database sjdbN=60508,   pGe.sjdbOverhang=149 
alignIntronMax=alignMatesGapMax=0, the max intron size will be approximately determined by    (2^winBinNbits)*winAnchorDistNbins=589824
Created thread # 1
 Thread #1 end of input stream, nextChar=-1
 Completed: thread #0
 Joined thread # 1

Can anyone help me ? Thank you very much :)

star rnaseq
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written by paxton • paxton

what's the number of reads in the input fastq files ?

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written by Pierre Lindenbaum Pierre Lindenbaum

Thanks for helping. I managed to fix the problem by using Docker.

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written by paxton • paxton

Check the input fastq files and share the command you have used for alignment.

#check the number of reads in the input fastq files
zgrep -c "^@" input_R1.fq.gz
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written by Arup Ghosh Arup Ghosh

1 answer

The line Thread #1 end of input stream, nextChar=-1 printed immediately after the genome loaded, with no reads in between, means STAR's read stream was empty from the very first byte -- the genome/index side is fine, nothing is arriving on the reads side. Check three things in order:

1. Are the FASTQs gzipped? If they are .gz and you did not pass --readFilesCommand zcat, that is by far the most common cause of exactly this Number of input reads | 0. STAR does not auto-detect compression; without the flag it cannot read a .gz and you get zero reads. Add:

--readFilesCommand zcat
# or: gunzip -c
# bgzipped: bgzip -cd

2. If --readFilesCommand is already set, test it standalone -- a wrong path or a silently-failing command produces an empty stream and the same symptom:

zcat sample_R1.fq.gz | head                        # should print FASTQ records
echo $(( $(zcat sample_R1.fq.gz | wc -l) / 4 ))    # read count

If that head is empty or errors, STAR is being handed nothing.

3. Double-check --readFilesIn (R1 then R2, space-separated; commas only for multiple lanes) and that you are not pointing at a trimmed/intermediate file that came out empty upstream.

If you paste the full STAR command (the --readFilesIn and --readFilesCommand lines especially), it will be obvious which of the three it is.

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written by SeqBench • SeqBench

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