Usual parameters? - quality string length is not equal to sequence length, fix your fastq file

[ERA]

Hello,

I run this command:

STAR --runThreadN 24  --genomeDir /genome_indexStar --readFilesCommand zcat --readFilesIn /read_1.fastq.gz /read_2.fastq.gz --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 16000000000 --quantMode TranscriptomeSAM --outSAMunmapped Within --outFilterType BySJout --outFileNamePrefix /read_

 

And I got this error message,

EXITING because of FATAL ERROR in reads input: quality string length is not equal to sequence length

@/home/leaf_23C_2/read.sra.27941168

GCCCTAGTCATCAGAAAAGGAAACCACTTGTGCCAGTTCCCACTCGCACCAGCTGAAAAACCAAGAACGTTTCCGACAGCCATCCATGAGCAGAATATTGC
SOLUTION: fix your fastq file

 

I have enough storage space in my working directory, and my reads are only 100nt long. And I think that no solution in this link https://groups.google.com/forum/#!topic/rna-star/vcNIAQScQt8 can be appropriate to resolve this problem. Any suggestion please?

 

Also, I want to run 2-pass mapping with re-generated genome based on the recommendation here (https://groups.google.com/forum/#!topic/rna-star/rBQK-ujtSh8). So I’d like to know the meaning of ‘usual’ (parameters) that is indicated in the 7.1 section (2-pass mapping with re-generated genome) of the STAR manual.

 

Thanks,

 

 

 

 

 

 

[Alexander Dobin]

Hi @ERA,

the error indicates the problem with the fastq file. Can you post a few lines around 

@/home/leaf_23C_2/read.sra.27941168 

from both read1 and read2 files. Is this the very first read in the file?

Cheers

Alex

[ERA]

Hi Alex,

This is the very last read of the files read_1.fastq and read_2.fastq 

$less read_1.fastq | grep -C 5 '@/home/leaf_23C_2/SRR1460550.sra.27941168 HWI-ST822:250:C2NH2ACXX:2:1304:10561:59086 length=101' | less

or $tail -n 5 read_1.fastq

CCCFFFFFHHHHHJJJJJIJJJJJJJIHJIJJJIJJJJJJJIJJJIIIIJJIGICGIIJCHIJJHHFFDEFEEEEEEDDDBDDDDBDDD<BDDEDDDDDD?

@/home/leaf_23C_2/SRR1460550.sra.27941167 HWI-ST822:250:C2NH2ACXX:2:1304:10535:59049 length=101

CAGAAGAAGCGTACTAGTAGACGTAGTTGCTCAAGAGCATTCATGGAATCCTCCTCCCCTGCTGCTACCCTGTCTCTCTCCCGTGTGTCATTGAAACATCA

+/home/leaf_23C_2/SRR1460550.sra.27941167 HWI-ST822:250:C2NH2ACXX:2:1304:10535:59049 length=101

CCCFFFFFDHHHFJJIEGGIJJI;GH@EHHGCGIEAHCGIGFGIG<BFF@?FGACFCFAHEHI@HGEEHCHBBDEBBCCE3>;?BABBC;;5>@ACDDDC>

@/home/leaf_23C_2/SRR1460550.sra.27941168 HWI-ST822:250:C2NH2ACXX:2:1304:10561:59086 length=101

GCCCTAGTCATCAGAAAAGGAAACCACTTGTGCCAGTTCCCACTCGCACCAGCTGAAAAACCAAGAACGTTTCCGACAGCCATCCATGAGCAGAATATTGC

+/wo

 

$less read_2.fastq | grep -C 5 '@/home/leaf_23C_2/SRR1460550.sra.27941168 HWI-ST822:250:C2NH2ACXX:2:1304:10561:59086 length=101' | less

or $tail -n 5 read_2.fastq

@BBFFFFFHHHHHJJJJJJJJJJJJJJJJJIJGGGHJJJJJJFHIIJJJHGHIJJJJJJHHHHHHFFFFFDDDDDDDDDDDDDBDDDDDDDDDDDDCDDD@

@/home/leaf_23C_2/SRR1460550.sra.27941167 HWI-ST822:250:C2NH2ACXX:2:1304:10535:59049 length=101

GATGTTTCAATGACACACGGGAGAGAGACAGGGCAGCAGCAGGGGAGGAGGATTCCATGAATGCTCTTGAGCAACTACGTCTACTAGTACGCTTCTTCTGA

+/home/leaf_23C_2//leaf_23C_2/SRR1460550.sra.27941167 HWI-ST822:250:C2NH2ACXX:2:1304:10535:59049 length=101

@@@DBBDB<CBFHHEFBEGDI:@AFF;GHAFGB)8@DGGHIIFFGBBECED7B;@BCDCCCDCCCCCC=ACCCCBC@CB?BCBCECC>CCBB?@9ACCC@#

@/home/leaf_23C_2/SRR1460550.sra.27941168 HWI-ST822:250:C2NH2ACXX:2:1304:10561:59086 length=101

AAGATATCGAGCTGCTTTTATTTTCATTTTTGGATTCTGGATGCTGGACCTTGCAAATAATACAGTTCAAGGACCTGCTCGTGTCCTCCTAGCTGATCTTT

+/wo

Best,

[Alexander Dobin]

Hi @ERA,

it looks like the last reads in both read1 and read2 miss two lines for: the header for the quality string (read id starting with +) and the quality string itself.

This explains the STAR's error message. If you delete the two lines from both files, it should work.

Cheers

Alex

[ERA]

Thank you Alex, the problem is fixed.

Cheers