How to use pre-alignments by bowtie2

Question

[Aki]

Hi, I read the paper (https://www.nature.com/articles/s41467-021-22720-0) using bowtie2 for ATAC-seq read alignment, and find the question about pre-alignments. Does anyone know how to use pre-alignments? I don't know how to use the output of pre-alignments by bowtie2 as an input file for alignments.

Following sentence is their protocol.

Bowtie2 was applied for pre-alignments to filter out reads that align to repetitive regions using “-k 1 -D 20 -R 3 -N 1 -L 20 -i S,1,0.50 -X 2000 –rg-id” parameters. For the remaining reads, Bowtie2 was used to map to GRCh38 with “–very-sensitive -X 2000–rg-id” options.

Thanks in advance

Answer 1

[GenoMax]

Unless bowtie2 allows piping directly (probably not) you may simply need to run round 1 of alignment. Followed by round2 of alignments with data you select from the first round.

[Aki]

Thanks, GenoMax. Do you know how to use the output of pre-alignments by bowtie2 as an input file for alignments like below? Or Should I use some package like SamToFastq and use this output for 2nd round bowtie2?

bowtie2 -k 1 -D 20 -R 3 -N 1 -L 20 -i S,1,0.50 -X 2000 --rg-id \
-x GRCm39 \
-1 pre_1.fq.gz \
-2 pre_2.fq.gz \
-S pre.sam

bowtie2 --very-sensitive -X 2000 --rg-id \
-x GRCm39 \
-1 pre.sam \
-S filtered.sam

[GenoMax]

Run exactly as shown above in two steps. Run first command to create pre.sam file, which is then used in the second command. So you will have to let command 1 complete before you start #2.

[Aki]

I got error at 2nd script. Could you suggest any idea?

Usage: 
  bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r> | --interleaved <i> | -b <bam>} [-S <sam>]

  <bt2-idx>  Index filename prefix (minus trailing .X.bt2).
             NOTE: Bowtie 1 and Bowtie 2 indexes are not compatible.
  <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).
  <i>        Files with interleaved paired-end FASTQ/FASTA reads
             Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
  <bam>      Files are unaligned BAM sorted by read name.
  <sam>      File for SAM output (default: stdout)

  <m1>, <m2>, <r> can be comma-separated lists (no whitespace) and can be
  specified many times.  E.g. '-U file1.fq,file2.fq -U file3.fq'.

Options (defaults in parentheses):

 Input:
  -q                 query input files are FASTQ .fq/.fastq (default)
  --tab5             query input files are TAB5 .tab5
  --tab6             query input files are TAB6 .tab6
  --qseq             query input files are in Illumina's qseq format
  -f                 query input files are (multi-)FASTA .fa/.mfa
  -r                 query input files are raw one-sequence-per-line
  -F k:<int>,i:<int> query input files are continuous FASTA where reads
                     are substrings (k-mers) extracted from a FASTA file <s>
                     and aligned at offsets 1, 1+i, 1+2i ... end of reference
  -c                 <m1>, <m2>, <r> are sequences themselves, not files
  -s/--skip <int>    skip the first <int> reads/pairs in the input (none)
  -u/--upto <int>    stop after first <int> reads/pairs (no limit)
  -5/--trim5 <int>   trim <int> bases from 5'/left end of reads (0)
  -3/--trim3 <int>   trim <int> bases from 3'/right end of reads (0)
  --trim-to [3:|5:]<int> trim reads exceeding <int> bases from either 3' or 5' end
                     If the read end is not specified then it defaults to 3 (0)
  --phred33          qualities are Phred+33 (default)
  --phred64          qualities are Phred+64
  --int-quals        qualities encoded as space-delimited integers

 Presets:                 Same as:
  For --end-to-end:
   --very-fast            -D 5 -R 1 -N 0 -L 22 -i S,0,2.50
   --fast                 -D 10 -R 2 -N 0 -L 22 -i S,0,2.50
   --sensitive            -D 15 -R 2 -N 0 -L 22 -i S,1,1.15 (default)
   --very-sensitive       -D 20 -R 3 -N 0 -L 20 -i S,1,0.50

  For --local:
   --very-fast-local      -D 5 -R 1 -N 0 -L 25 -i S,1,2.00
   --fast-local           -D 10 -R 2 -N 0 -L 22 -i S,1,1.75
   --sensitive-local      -D 15 -R 2 -N 0 -L 20 -i S,1,0.75 (default)
   --very-sensitive-local -D 20 -R 3 -N 0 -L 20 -i S,1,0.50

 Alignment:
  -N <int>           max # mismatches in seed alignment; can be 0 or 1 (0)
  -L <int>           length of seed substrings; must be >3, <32 (22)
  -i <func>          interval between seed substrings w/r/t read len (S,1,1.15)
  --n-ceil <func>    func for max # non-A/C/G/Ts permitted in aln (L,0,0.15)
  --dpad <int>       include <int> extra ref chars on sides of DP table (15)
  --gbar <int>       disallow gaps within <int> nucs of read extremes (4)
  --ignore-quals     treat all quality values as 30 on Phred scale (off)
  --nofw             do not align forward (original) version of read (off)
  --norc             do not align reverse-complement version of read (off)
  --no-1mm-upfront   do not allow 1 mismatch alignments before attempting to
                     scan for the optimal seeded alignments
  --end-to-end       entire read must align; no clipping (on)
   OR
  --local            local alignment; ends might be soft clipped (off)

 Scoring:
  --ma <int>         match bonus (0 for --end-to-end, 2 for --local) 
  --mp <int>         max penalty for mismatch; lower qual = lower penalty (6)
  --np <int>         penalty for non-A/C/G/Ts in read/ref (1)
  --rdg <int>,<int>  read gap open, extend penalties (5,3)
  --rfg <int>,<int>  reference gap open, extend penalties (5,3)
  --score-min <func> min acceptable alignment score w/r/t read length
                     (G,20,8 for local, L,-0.6,-0.6 for end-to-end)

 Reporting:
  (default)          look for multiple alignments, report best, with MAPQ
   OR
  -k <int>           report up to <int> alns per read; MAPQ not meaningful
   OR
  -a/--all           report all alignments; very slow, MAPQ not meaningful

 Effort:
  -D <int>           give up extending after <int> failed extends in a row (15)
  -R <int>           for reads w/ repetitive seeds, try <int> sets of seeds (2)

 Paired-end:
  -I/--minins <int>  minimum fragment length (0)
  -X/--maxins <int>  maximum fragment length (500)
  --fr/--rf/--ff     -1, -2 mates align fw/rev, rev/fw, fw/fw (--fr)
  --no-mixed         suppress unpaired alignments for paired reads
  --no-discordant    suppress discordant alignments for paired reads
  --dovetail         concordant when mates extend past each other
  --no-contain       not concordant when one mate alignment contains other
  --no-overlap       not concordant when mates overlap at all

 BAM:
  --align-paired-reads
                     Bowtie2 will, by default, attempt to align unpaired BAM reads.
                     Use this option to align paired-end reads instead.
  --preserve-tags    Preserve tags from the original BAM record by
                     appending them to the end of the corresponding SAM output.

 Output:
  -t/--time          print wall-clock time taken by search phases
  --un <path>        write unpaired reads that didn't align to <path>
  --al <path>        write unpaired reads that aligned at least once to <path>
  --un-conc <path>   write pairs that didn't align concordantly to <path>
  --al-conc <path>   write pairs that aligned concordantly at least once to <path>
    (Note: for --un, --al, --un-conc, or --al-conc, add '-gz' to the option name, e.g.
    --un-gz <path>, to gzip compress output, or add '-bz2' to bzip2 compress output.)
  --quiet            print nothing to stderr except serious errors
  --met-file <path>  send metrics to file at <path> (off)
  --met-stderr       send metrics to stderr (off)
  --met <int>        report internal counters & metrics every <int> secs (1)
  --no-unal          suppress SAM records for unaligned reads
  --no-head          suppress header lines, i.e. lines starting with @
  --no-sq            suppress @SQ header lines
  --rg-id <text>     set read group id, reflected in @RG line and RG:Z: opt field
  --rg <text>        add <text> ("lab:value") to @RG line of SAM header.
                     Note: @RG line only printed when --rg-id is set.
  --omit-sec-seq     put '*' in SEQ and QUAL fields for secondary alignments.
  --sam-no-qname-trunc
                     Suppress standard behavior of truncating readname at first whitespace 
                     at the expense of generating non-standard SAM.
  --xeq              Use '='/'X', instead of 'M,' to specify matches/mismatches in SAM record.
  --soft-clipped-unmapped-tlen
                     Exclude soft-clipped bases when reporting TLEN
  --sam-append-comment
                     Append FASTA/FASTQ comment to SAM record

 Performance:
  -p/--threads <int> number of alignment threads to launch (1)
  --reorder          force SAM output order to match order of input reads
  --mm               use memory-mapped I/O for index; many 'bowtie's can share

 Other:
  --qc-filter        filter out reads that are bad according to QSEQ filter
  --seed <int>       seed for random number generator (0)
  --non-deterministic
                     seed rand. gen. arbitrarily instead of using read attributes
  --version          print version information and quit
  -h/--help          print this usage message
***
Error: Must specify at least one read input with -U/-1/-2
(ERR): bowtie2-align exited with value 1