Odd error with genome preparation command

[jws777]

I have been trying to align some files to the mouse genome but when I come to the genome preparation step, I get this error (sorry there is so much nonsense):

Bismark Genome Preparation - Step II: Bisulfite converting reference genome

conversions performed: chromosome C->T G->A The specified chromosome ( ˎ,ɶ6l*NB@2{4 $:/Qi;<soo4<VU͚w;E??O???_/g0000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000Bowtie 2 seems to be working fine (tested command 'bowtie2 --version' [2.3.5]) Output format is BAM (default)

The genome file I downloaded from ENSEMBL is Mus_musculus.GRCm39.dna.toplevel.fa

This is the code I am running:

verify which other packages available

which bowtie2 which samtools which fastqc which bismark

Run fastQC on .fastq files

fastqc SRR691421_RRBS.fastq

Quality and adapter trimming

trim_galore SRR691421_RRBS.fastq

Preparation for alignment with Bismark

bismark_genome_preparation --verbose /mnt/c/Users/my_lab/Documents/my_name/Beerman_et_al_raw_data/genome/

(And the .fa file is definitely in the folder specified). The QC and trimming all runs fine before this step.

Any help very much appreciated, thank you very much!

[FelixKrueger]

There are some weird things going on. Maybe first of all, I am not sure if the TOPLEVEL file is generally a good choice, as it may contain haplotype and patch regions (which may lead to ambiguous alignments, and therefore result in sequences getting excluded).

---------
TOPLEVEL
---------
These files contains all sequence regions flagged as toplevel in an Ensembl
schema. This includes chromsomes, regions not assembled into chromosomes and
N padded haplotype/patch regions.

-----------------
PRIMARY ASSEMBLY
-----------------
Primary assembly contains all toplevel sequence regions excluding haplotypes
and patches. This file is best used for performing sequence similarity searches
where patch and haplotype sequences would confuse analysis. If the primary
assembly file is not present, that indicates that there are no haplotype/patch
regions, and the 'toplevel' file is equivalent.

For the mouse genome, I think they are both equivalent though, and both should work equally well.

On my end, using Bismark v0.23.0, Bowtie2 v2.4.2, and Samtools v1.11, the genome prep works just fine:

Bismark Genome Preparation - Step II: Bisulfite converting reference genome

conversions performed:
chromosome  C->T    G->A
1   39505587    39475953
2   37509143    37549551
3   31632630    31689758
4   32273771    32299184
5   31474546    31480386
6   30324207    30303520
7   30522166    30486722
8   26636031    26613974
9   25887273    25865951
10  26297538    26349838
11  26029159    26024038
12  24373800    24425370
13  24404130    24399021
14  24926453    24936665
15  21183326    21165928
16  19408269    19424734
17  19612158    19591811
18  18129221    18175890
19  12452755    12424366
X   32224667    32254982
Y   17244325    17161729
MT  3976    2013
JH584299.1  196679  194475
GL456233.2  96662   95939
JH584301.1  49630   52220
GL456211.1  53784   52384
GL456221.1  44248   45898
JH584297.1  42059   42163
JH584296.1  41022   40705
GL456354.1  40330   40258
JH584298.1  37547   38061
JH584300.1  35488   36307
GL456219.1  34883   35727
GL456210.1  37533   37162
JH584303.1  31726   31235
JH584302.1  30031   30742
GL456212.1  33862   33317
JH584304.1  26005   22257
GL456379.1  13182   13573
GL456366.1  8411    8579
GL456367.1  7202    8000
GL456239.1  8413    8616
GL456383.1  6954    4475
GL456385.1  6854    6900
GL456360.1  6402    6234
GL456378.1  6106    6240
MU069435.1  6707    6277
GL456389.1  5331    5344
GL456372.1  5466    5079
GL456370.1  3287    2675
GL456381.1  4370    5498
GL456387.1  4791    5863
GL456390.1  1930    2970
GL456394.1  4156    4651
GL456392.1  3685    4929
GL456382.1  4209    4285
GL456359.1  4277    4578
GL456396.1  3855    4663
GL456368.1  3755    3723
MU069434.1  2091    1960
JH584295.1  611 611

Total number of conversions performed:
C->T:   553008665
G->A:   553055957

So I suspect that it something really odd, such as the version of gunzip on your system, or maybe the format of the incoming FastA file is somehow corrupt? Which platform are you running this on? Here are the commands I used:

wget http://ftp.ensembl.org/pub/release-104/fasta/mus_musculus/dna/Mus_musculus.GRCm39.dna.toplevel.fa.gz
file Mus_musculus.GRCm39.dna.toplevel.fq.gz
Mus_musculus.GRCm39.dna.toplevel.fq.gz: gzip compressed data

(maybe you have some really odd file endings, and require dos2unix or mac2unix before running the command again?) and this was my Bismark indexing command:

bismark_genome_preparation . --verbose

Maybe it would be worth looking at the file endings first, then update potentially outdated software packages?

[jws777]

Thank you Felix - I had manually downloaded the genome file, unzipped it, and added it to the folder. I don't know why it would be different but when I did it using your commands then it worked perfectly and I have the same results as you showed above. Thanks so much for your quick response!

[FelixKrueger]

Excellent, I am glad it's all fine now. All the best