[E::bwa_idx_load_from_disk] fail to locate the index files

Question

[jan.bartel90]

Hello,

I am facing a problem unfortunately I cannot solve. I want to use padlock primers for DNA methylation analysis according to this protocol -> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461232/

For sequence data analysis I tried to use the software written and published by the corresponding authors -> https://github.com/dinhdiep/BisReadMapper

For alignment the analysis software implements BWA and you have to give the software the path where to find BWA which I did on our server.

Furthermore I created the index files according to the description with bwa and samtools. "Next, use '''bwa''' to create the index files from the reference sequences (i. e. genome.fa file) for alignment. Then, use '''samtools''' to quickly generate the genome index file from the reference sequences." and stored them in a variety of different directions.

Unfortunately BisReadMapper aborts its operations and displays these notes:

Multicore support not enabled. Proceeding with single-core trimming.

Path to Cutadapt set as: 'cutadapt' (default)

Cutadapt seems to be working fine (tested command 'cutadapt --version')

Cutadapt version: 1.15

single-core operation.

[MasterBisReadMapper] Begins BisReadMapper...

[M::bwa_idx_load_from_disk] read 0 ALT contigs

[M::process] read 362328 sequences (40000119 bp)...

[M::process] read 67887 sequences (7609855 bp)...

[M::mem_process_seqs] Processed 362328 reads in 552.000 CPU sec, 138.088 real sec

[M::mem_process_seqs] Processed 67887 reads in 153.356 CPU sec, 38.214 real sec

[main] Version: 0.7.17-r1188

[main] CMD: /misc/software/ngs/bwa/src/bwa-0.7.17/bwa mem -t 4 -B2 -c 1000 /misc/software/ngs/bisreadmapper/src/2019-12-09/BisReadMapper/src/mm10.fa.bis.CT PadlockV1_map/r0.Padlock-V1-full-meth_S1_L001_R1_001.PE.1.encoded

[main] Real time: 219.591 sec; CPU: 713.764 sec

[E::bwa_idx_load_from_disk] fail to locate the index files
system problem (exit 256): 

[MasterBisReadMapper] Failed to run BisReadMapper (exit 256): 
Command used:
    /misc/software/ngs/bisreadmapper/src/2019-12-09/BisReadMapper/src/BisReadMapper.pl -r PadlockV1_map/Padlock-V1-full-meth_S1_L001_R1_001_val_1.fq -W /misc/software/ngs/bisreadmapper/src/2019-12-09/BisReadMapper/src/mm10.fa.bis.CT -C /misc/software/ngs/bisreadmapper/src/2019-12-09/BisReadMapper/src/mm10.fa.bis.GA -g /misc/software/ngs/bisreadmapper/src/2019-12-09/BisReadMapper/src/mm10.fa.fai -a /misc/software/ngs/bwa/src/bwa-0.7.17/bwa -b 33 -p 4 -n PadlockV1_map/r0.Padlock-V1-full-meth_S1_L001_R1_001.PE > PadlockV1_map/r0.Padlock-V1-full-meth_S1_L001_R1_001.PE.status
[MasterBisReadMapper] !!!! Error while processing Padlock-V1-full-meth_S1_L001_R1_001.fastq,Padlock-V1-full-meth_S1_L001_R2_001.fastq in /misc/software/ngs/bisreadmapper/src/2019-12-09/BisReadMapper/src/Reads !!!!

[MasterBisReadMapper] All reads have been mapped

Is there anything I did wrong or can someone help me with the issue?

software version is bwa-0.7.17

Thank you very much and all the best,

Jan

[Pierre Lindenbaum]

/misc/software/ngs/bisreadmapper/src/2019-12-09/BisReadMapper/src/mm10.fa.bis.CT doesn't look like a fasta file (may be it is, but what a strange suffix....)

[ATpoint]

Did you create an index based on the in-silico bisulfite-converted genome? If not then please read the documentation. This must be done and I guess this is the index missing here.

[jan.bartel90]

hello,

thank u for your answer. So i in-silico bisulfite converted my reference genome (in this case mm10.fa) with the provided script genomePrep.pl and it gives me these two files mm10.fa.bis.CT & mm10.fa.GA. These two files are then given to MasterBisReadMapper.pl in a textfile providing all the necessary paths. Furthermore I created the index of mm10.fa with bwa and samtools given me these outputs: mm10.fa.amb, mm10.fa.ann, mm10.fa.bwt, mm10.fa.pac, mm10.fa.sa and mm10.fa.fai. I did not run samtools index command on the in-silico bisulfite converted reference because the program just does not let me do that giving me errors when I want to run samtools faidx in either fa.bis.CT or fa.bis.GA.

Thank u,

Jan

[N15]

can you share your index command?

[jan.bartel90]

yes,

its

bwa index mm10.fa

and

samtools faidx mm10.fa

Thank U,

Jan