TGS-GapCloser

A gap-closing software tool that uses error-prone long reads generated by third-generation-sequence techniques (Pacbio, Oxford Nanopore, etc.) or preassembled contigs to fill N-gap in the genome assembly.

• Both raw reads and pre-error-corrected reads are acceptable as input.

• If only raw long reads are provided, it polishes raw TGS reads by calling Racon.

• If additional NGS short reads are available, it polishes raw TGS reads by calling Pilon.

Note: only fasta format of TGS reads is acceptable.

Citing TGS-GapCloser

If you use TGS-GapCloser in your work, please cite: TGS-GapCloser: A fast and accurate gap closer for large genomes with low coverage of error-prone long reads Mengyang Xu, Lidong Guo, Shengqiang Gu, Ou Wang, Rui Zhang, Brock A Peters, Guangyi Fan, Xin Liu, Xun Xu, Li Deng, Yongwei Zhang GigaScience, Volume 9, Issue 9, 1 September 2020, giaa094, https://doi.org/10.1093/gigascience/giaa094

Dependencies

1. gcc 4.4+
2. make 3.8+
3. minimap2

Installation

Download

git clone  https://github.com/BGI-Qingdao/TGS-GapCloser.git YOUR-INSTALL-DIR

Compile

configure minimap2

• If you have already installed a minimap2, then link it here

rm -rf YOUR-INSTALL-DIR/minimap2
ln -s MINIMAP2-PATH YOUR-INSTALL-DIR/

• Otherwise, you need to download it by:

cd YOUR-INSTALL-DIR
git submodule init
git submodule update

compile main src

cd YOUR-INSTALL-DIR
make

Conda install

not available

if you install by conda, please install minimap2 first and make sure that minimap2 is available in your environment.

Usage

Usage:
      tgsgapcloser2 --scaff SCAFF_FILE --reads TGS_READS_FILE --output OUT_PREFIX [options...]
      required:
          --scaff     <draft scaffolds>    input draft scaffolds.
          --reads     <TGS reads>          input TGS reads.
          --output    <output prefix>      output prefix.
     ## error correction module
          --ne                             do not correct errors. by default.
          or
          --racon     <racon>              installed racon path. Can be installed following https://github.com/isovic/racon
          or
          --pilon     <pilon>              pilon jar package. Can be downloaded from https://github.com/broadinstitute/pilon/releases/download/v1.23/pilon-1.23.jar
          --java      <java>               installed java path.
          --ngs       <ngs_reads>          input NGS reads used for pilon.
          --samtools  <samtools>           installed samtools path.
          
          
      optional:

          --minmap_arg <minmap2 args>      for example, --minmap_arg \' -x ava-ont\'
                                           arg must be wrapped by \' \'
          --tgstype   <pb/ont/hifi>        TGS type. ont by default.
          --min_idy   <float>              minimum identity for filtering candidate TGS sequences.
                                           0.3 for ont by default.
                                           0.2 for pb/hifi by default.
          --min_match <int>                minimum matched length for filtering candidate TGS sequences.
                                           300 for ont by default.
                                           200 for pb/hifi by default.
          --thread    <int>                number of threads uesd. 16 by default.
          --pilon_mem <int>                memory used for pilon, passing to -Xmx.  can use “m” or “M” for MB, or “g” or “G” for GB. 300G by default.
          --chunk     <int>                split candidates into # of chunks to separately correct errors. 3 by default.
          --p_round   <int>                iteration # of error corretion by pilon. 3 by default.
          --r_round   <int>                iteration # of error corretion by racon. 3 by default.
          --g_check                        gap size diff check. off by default.
          --min_nread <int>                minimum number of reads that can bridge this gap. 1 by default.
          --max_nread <int>                maximum number of reads that can bridge this gap. -1 by default.
          --max_candidate <int>            maximum number of candidate alignments used for error correction and gap filling. 200 by default.

WARNING: only fasta format TGS reads is supported and fastq format will lead to program crashing !

Examples

an example of pre-corrected TGS reads without error correction

YOUR-INSTALL-DIR/tgsgapcloser  \
        --scaff  scaffold-path/scaffold.fasta \
        --reads  tgs-reads-path/tgs.reads.fasta \
        --output test_ne  \
        --ne \
        >pipe.log 2>pipe.err

An example of raw ONT reads with error correction using long reads only

YOUR-INSTALL-DIR/tgsgapcloser  \
        --scaff  scaffold-path/scaffold.fasta \
        --reads  tgs-reads-path/tgs.reads.fasta \
        --output test_racon \
        --racon  racon-path/bin/racon \
        >pipe.log 2>pipe.err

An example of raw ONT reads with error correction using NGS reads

YOUR-INSTALL-DIR/tgsgapcloser  \
        --scaff  scaffold-path/scaffold.fasta \
        --reads  tgs-reads-path/tgs.reads.fasta \
        --output test_pilon \
        --pilon  pilon-path/pilon-1.23.jar  \
        --ngs    ngs-reads-path/ngs.reads.fastq.gz  \
        --samtools samtools-path/bin/samtools  \
        --java    java-path/bin/java \
        >pipe.log 2>pipe.err

Using Pacbio reads

• default TGS type is ONT, use --tgstype to change it .

    --tgstype ont
    to 
    --tgstype pb
or
    to 
    --tgstype hifi

• an example of raw Pacbio reads with error correction using long reads only

YOUR-INSTALL-DIR/tgsgapcloser  \
        --scaff  scaffold-path/scaffold.fasta \
        --reads  tgs-reads-path/tgs.reads.fasta \
        --output test_racon \
        --racon  raconn-path/bin/racon \
        --tgstype pb \
        >pipe.log 2>pipe.err

When you need to appoint custom minimap2 parameters

Use --minmap_arg ' your-own minimap2 args'

This is useful when you want to avoid a huge paf file.

for example, if you use HiFi Reads, you may try --minmap_arg '-x asm20'

YOUR-INSTALL-DIR/tgsgapcloser  \
        --scaff  scaffold-path/scaffold.fasta \
        --reads  tgs-reads-path/tgs.reads.fasta \
        --output test_racon \
        --minmap_arg '-x asm20' \
        --racon  raconn-path/bin/racon \
        --tgstype pb \
        >pipe.log 2>pipe.err

Output

• your-prefix.scaff_seq
  • this is the final assembly after gap filling
• your-prefix.gap_fill_details
  • details about how the final assembly was assembled

format of your-prefix.gap_fill_details

an example:

>scaffold_1
1	1000	S	1000	2000
1001	1010	N
1011	1100	S	2201	2290
1101	1110	F
1111	1200	S	2301	2390
>scaffold_2
......

detailed information

1. each scaffold name is followed by its data lines.
2. a data line consists of 3 or 5 columns and describes the source of each segment in the final sequence:
   • column 1 is the segment's first bp position in the final sequence.
   • column 2 is the segment's last bp position in the final sequence.
   • column 3 is the segment's type, 'S', 'N', or 'F'.
     • 'S' means this segment is a segment of the input sequence and this line includes other two columns:
       • column 4 is the segment's first bp position in the input sequence.
       • column 5 is the segment's last bp position in the input sequence.
     • 'N' means this segment is an N area.
     • 'F' means this segment is a filled sequence from TGS reads.

Contact

Any questions, please feel free to ask [email protected] or [email protected]

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