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I need help with bowtie2 to map reads different length

Hi, I need help mapping Paired-end samples. My samples were filtered by Prinseq. But after the filter, this error appears:

Error, fewer reads in file specified with -2 than in file specified with -1
terminate called after throwing an instance of 'int'
Aborted (core dumped)
(ERR): bowtie2-align exited with value 134

I used following command:

bowtie2 \
  --very-sensitive \
  -x 
/home/douglas_junior_lbcm/reference/S288C_reference_Modificado_index_base \
  -1 /home/douglas_junior_lbcm/analise_qualidade/amostras_filtradas/240_1_prinseq_filtered_q33_30.fastq \
  -2 
/home/douglas_junior_lbcm/analise_qualidade/amostras_filtradas/240_2_prinseq_filtered_q33_30.fastq \
  -S 240_ParInf_Q3330f_S288C_bowtie2.sam

Anyone can help me?

bowtie2
• 1,684 views • link
updated by Ram Ram • written by DByter • DByter

1 answer 

 wc  -l /home/douglas_junior_lbcm/analise_qualidade/amostras_filtradas/240_1_prinseq_filtered_q33_30.fastq /home/douglas_junior_lbcm/analise_qualidade/amostras_filtradas/240_2_prinseq_filtered_q33_30.fastq

should return the same number of lines for both files. Otherwise, this is a problem with the command that generated the fastq files.

• 0 views • link
written by Pierre Lindenbaum Pierre Lindenbaum 
26180292 /home/douglas_junior_lbcm/analise_qualidade/amostras_filtradas/240_1_prinseq_filtered_q33_30.fastq
26065868 /home/douglas_junior_lbcm/analise_qualidade/amostras_filtradas/240_2_prinseq_filtered_q33_30.fastq
52246160 total

This is the result.

The difference occurred after filtering. Do you have any suggestions on how to avoid this problem even using the same filtering parameters?

I tried to find some function to ignore different sizes of reads and I couldn't find it.

• 0 views • link
updated by Ram Ram • written by DByter • DByter

You need to use a preprocessing tool that keeps files in sync. Trimmomatic, fastp, cutadapt, to name a few.

• 0 views • link
written by ATpoint ATpoint

pelo fastqc eu nao conseguiria realizar esse procedimento então? (Google Translate: "by fastqc I would not be able to perform this procedure then?")

I used following command to forward:

prinseq-lite \
  -verbose \
  -fastq /home/douglas_junior_lbcm/analise_qualidade/amostras_brutas/NG-21420_Samplen240_1.fastq \
  -out_good /home/douglas_junior_lbcm/analise_qualidade/amostras_filtradas/teste/240_1_prinseq_filtered_q33_30 \
  -trim_qual_right 33 \
  -trim_qual_window 5 \
  -trim_qual_step 1 \
  -trim_qual_type min \
  -min_len 30 \
  -out_format 3

to reverse:

prinseq-lite \
  -verbose \
  -fastq /home/douglas_junior_lbcm/analise_qualidade/amostras_brutas/NG-21420_Samplen240_1.fastq \
  -out_good /home/douglas_junior_lbcm/analise_qualidade/amostras_filtradas/teste/240_2_prinseq_filtered_q33_30 \
  -trim_qual_right 33 \
  -trim_qual_window 5 \
  -trim_qual_step 1 \
  -trim_qual_type min \
  -min_len 30 \
  -out_format 3
• 0 views • link
updated by Ram Ram • written by DByter • DByter

Both your commands seem to work on the same FASTQ file. Is that a copy-paste error?

• 0 views • link
written by Ram Ram

I think the point is that this tool processes one file at a time and hence does not keeps them in sync. I gave above suggestions for alternative tools. No, fastqc cannot do any of that, it's an analysis program that "only" collects some stats.

• 0 views • link
written by ATpoint ATpoint

OP is using prinseq-lite, which has a -fastq <R1> -fastq2 <R2> option for PE data. OP does not seem to pick up on the idea of processing both files "in sync" as you call it. And you're right, FASTQC's inability to process both ends together is not a problem, as nothing that FASTQC does really depends on it being able to do that.

• 0 views • link
written by Ram Ram

Then use that option @OP :)

• 0 views • link
updated by Ram Ram • written by ATpoint ATpoint

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