Hisat2 after the use of trimmomatic software: (ERR): hisat2-align exited with value 134

Question

[Mithil Gaikwad]

I have used trimmomatic software for trimming my paired-end RNA-Seq data, Now I have four output results viz.: sample_1p.fq.gz, sample_1u.fq.gz, sample_2p.fq.gz, and sample_2u.fq.gz. I followed this Question Which suggests using both samples paired and unpaired separately for alignment and then merge it using samtools

Then I did alignment procedure for paired end first,

hisat2 -p 8 --dta -x "/path/to/Index_files/indexfile" -1 sample_1p.fastq.gz -2 sample_2p.fastq.gz -S sample_paired.sam

resulted as

8763987 reads; of these:
  8763987 (100.00%) were paired; of these:
    1431464 (16.33%) aligned concordantly 0 times
    7029760 (80.21%) aligned concordantly exactly 1 time
    302763 (3.45%) aligned concordantly >1 times
    ----
    1431464 pairs aligned concordantly 0 times; of these:
      60100 (4.20%) aligned discordantly 1 time
    ----
    1371364 pairs aligned 0 times concordantly or discordantly; of these:
      2742728 mates make up the pairs; of these:
        1504242 (54.84%) aligned 0 times
        1055477 (38.48%) aligned exactly 1 time
        183009 (6.67%) aligned >1 times
91.42% overall alignment rate

and then for unpaired sample,

hisat2 -p 8 --dta -x "/path/to/Index_files/indexfile" -1 sample_1u.fastq.gz -2 sample_2u.fastq.gz -S sample_unpaired.sam

which resulted as

Error, fewer reads in the file specified with -2 than in file specified with -1 terminate called after throwing an instance of 'int' Aborted (core dumped) (ERR): hisat2-align exited with value 134

Now since my sample reads after trimming are not equal in both of the samples. My question: Is it fine If I only use paired sample data for alignment, quantification, and downstream Differential gene expression analysis? Are unpaired reads important for downstream processing? Or my trimming was more aggressive which retain unequal no. of reads?

Any help in this regard will be deeply appreciated. Thank you in advance.

Answer 1

[ATpoint]

From the manual:

Usage: 
  hisat2 [options]* -x <ht2-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <sam>]

  <ht2-idx>  Index filename prefix (minus trailing .X.ht2).
  <m1>       Files with #1 mates, paired with files in <m2>.
             Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
  <m2>       Files with #2 mates, paired with files in <m1>.
             Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
  <r>        Files with unpaired reads.
             Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
  <SRA accession number>        Comma-separated list of SRA accession numbers, e.g. --sra-acc SRR353653,SRR353654.
  <sam>      File for SAM output (default: stdout)

If files are unpaired, use -U, not -1/2