Seqanswers Leaderboard Ad



No announcement yet.
  • Filter
  • Time
  • Show
Clear All
new posts

  • GATK RNA-Seq pathway Split 'n' Trim- problems with .bai and .fai index compatibility

    I am processing aligned RNA-Seq data to get phased haplotypes of novel genes in sea stars. I am working through the analysis pipeline that used the Genome Analysis Toolkit suite of software, and is detailed on the Broad Institute website:


    I am up to the Split 'n' Trim stage of the pipeline, where the SplitNCigarReads tool is used to refine the sequence alignment. I have hit some problems with my index files. The order of the scaffolds and contigs for .fai (fasta index file) and .bai (index for bam file; bam file has been sorted, read-groups have been added and duplicated marked) files is different.

    e.g. the .fai file:
    Scaffold1 154793 11 70 71
    Scaffold2 383464 157027 70 71
    Scaffold3 336159 545981 70 71

    eg. the .bai file (samtools idxstats filename.bam):
    Scaffold1 154793 0 0
    Scaffold10 244803 0 0
    Scaffold100 200181 0 0

    I therefore got the following error from GATK:
    ##### ERROR MESSAGE: Input files reads and reference have incompatible contigs: Relative ordering of overlapping contigs differs, which is unsafe.
    ##### ERROR reads contigs = [Scaffold1, Scaffold10, Scaffold100, Scaffold1000...
    ##### ERROR reference contigs = [Scaffold1, Scaffold2, Scaffold3, Scaffold4…

    I wrote a script to change the order of the items in the .fai file so they would be in the same order as the .bai file, but this produced another error:
    ##### ERROR MESSAGE: Couldn't read file filename.fa because Mismatch between sequence dictionary fasta index for filename.fa, sequence 'Scaffold2' != 'Scaffold10'.

    Can anyone suggest a good work around for this problem? Do I need to change the order of entries in the fasta file reference sequence? Should I alter the .dict file (scaffolds/contigs are in the same order as they are in the original .fai file)?
    Last edited by gwilymh; 11-17-2014, 06:03 PM. Reason: Specifying the specific part of the pipeline where I had the problem

  • #2
    You can't just reorder the lines in the fai file, they need to be in the exact same order as the contigs in the fasta file.

    You have two options:
    (1) Reorder the fasta file. If there are a LOT of contigs then this could be a pain. The simplest method is to split the fasta file by chromosome and then reconcatenate the proper order.
    (2) Reorder the BAM file. You can use picard tool's ReorderSam command to do this. This will order things to match the fasta file.

    I expect option (2) is easier in your case.


    Latest Articles


    • seqadmin
      Exploring the Dynamics of the Tumor Microenvironment
      by seqadmin

      The complexity of cancer is clearly demonstrated in the diverse ecosystem of the tumor microenvironment (TME). The TME is made up of numerous cell types and its development begins with the changes that happen during oncogenesis. “Genomic mutations, copy number changes, epigenetic alterations, and alternative gene expression occur to varying degrees within the affected tumor cells,” explained Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta. “As...
      07-08-2024, 03:19 PM
    • seqadmin
      Exploring Human Diversity Through Large-Scale Omics
      by seqadmin

      In 2003, researchers from the Human Genome Project (HGP) announced the most comprehensive genome to date1. Although the genome wasn’t fully completed until nearly 20 years later2, numerous large-scale projects, such as the International HapMap Project and 1000 Genomes Project, continued the HGP's work, capturing extensive variation and genomic diversity within humans. Recently, newer initiatives have significantly increased in scale and expanded beyond genomics, offering a more detailed...
      06-25-2024, 06:43 AM





    Topics Statistics Last Post
    Started by seqadmin, 07-19-2024, 07:20 AM
    0 responses
    Last Post seqadmin  
    Started by seqadmin, 07-16-2024, 05:49 AM
    0 responses
    Last Post seqadmin  
    Started by seqadmin, 07-15-2024, 06:53 AM
    0 responses
    Last Post seqadmin  
    Started by seqadmin, 07-10-2024, 07:30 AM
    0 responses
    Last Post seqadmin