no spikeIn chromosomes found with extention _spikein

[sunta3iouxos]

Hi Katarzyna and everyone else,
I can not run the spikeIns strings for Chip-seq, this is a CUT&RUN protocol that has the bacteria cary over that can be used to "normalize the samples".
this is the full error:

ChIP-seq -d /mnt/c/AP01/bamSpikes -j 8 --local --useSpikeInForNorm --getSizeFactorsFrom genome --peakCaller Genrich --sampleSheet /mnt/c/AP01/bamSpikes/H3K36me3.tsv --windowSize 500 mm10_gencodeM19_spikes /mnt/c/AP01/bamSpikes/H3K36me3_chip_ty
pe.yaml
Sample sheet found and header is ok!

---- This analysis has been done using snakePipes version 2.7.3 ----
Sample sheet found and header is ok!
SystemExit in line 256 of mambaforge/envs/snakePipes/lib/python3.11/site-packages/snakePipes/workflows/ChIP-seq/internals.snakefile:
1
  File "mambaforge/envs/snakePipes/lib/python3.11/site-packages/snakePipes/workflows/ChIP-seq/Snakefile", line 22, in <module>
  File "mambaforge/envs/snakePipes/lib/python3.11/site-packages/snakePipes/workflows/ChIP-seq/internals.snakefile", line 256, in <module>
  File "<frozen _sitebuiltins>", line 26, in __call__

 No spikein genome detected - no spikeIn chromosomes found with extention _spikein .


Error: snakemake returned an error code of 1, so processing is incomplete!

The steps I have followed:

createIndices -o CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes --tools bowtie2 -j 6 --local --genomeURL CUT-RUNTools-2.0/assemblies/GRCm38_gencode_release19/genome_fasta/genome.fa --gtfURL CUT-RUNTools-2.0/assemblies/GRCm38_gencode_release19/annotation/genes.gtf  --spikeinGenomeURL CUT-RUNTools-2.0/assemblies/EB1/Sequence/WholeGenomeFasta/genome.fa --spikeinGtfURL CUT-RUNTools-2.0/assemblies/EB1/Annotation/Genes/genes.gtf  --blacklist CUT-RUNTools-2.0/blacklist/mm10.blacklist_merged.bed  mm10_gencodeM19_spikes
DNA-mapping -i /mnt/c/AP01/ -o /mnt/c/AP01/bamSpikes --local --trim --trimmer fastp --trimmerOptions "--trim_poly_g --trim_poly_x -Q -L --correction" --dedup --mapq 2 -j 8 mm10_gencodeM19_spikes
ChIP-seq -d /mnt/c/AP01/bamSpikes -j 8 --local --useSpikeInForNorm --getSizeFactorsFrom genome --peakCaller Genrich --sampleSheet /mnt/c/AP01/bamSpikes/H3K36me3.tsv --windowSize 500 mm10_gencodeM19_spike /mnt/c/AP01/bamSpikes/H3K36me3_chip_type.yaml

and this is my hybrid genome yalm
mambaforge/envs/snakePipes/lib/python3.11/site-packages/snakePipes/shared/organisms/mm10_gencodeM19_spikes.yaml

blacklist_bed: CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/annotation/blacklist.bed
bowtie2_index: CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/BowtieIndex/genome
bwa_index: ''
bwa_mem2_index: ''
bwameth2_index: ''
bwameth_index: ''
extended_coding_regions_gtf: CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/annotation/genes.slop.gtf
genes_bed: CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/annotation/genes.bed
genes_gtf: CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/annotation/genes.gtf
genome_2bit: CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/genome_fasta/genome.2bit
genome_dict: CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/genome_fasta/genome.dict
genome_fasta: CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/genome_fasta/genome.fa
genome_index: CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/genome_fasta/genome.fa.fai
genome_size: 2652783500
hisat2_index: ''
ignoreForNormalization: ''
known_splicesites: ''
rmsk_file: ''
spikein_blacklist_bed: ''
spikein_genes_gtf: CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/annotation/spikein_genes.gtf
star_index: ''

indexed genome:

genome.1.bt2  genome.2.bt2  genome.3.bt2  genome.4.bt2  genome.fa  genome.rev.1.bt2  genome.rev.2.bt2

[sunta3iouxos]

just to add that the bam file header is:
the added E.coli chromosome is named Chromosome. Do I need to add this one in the --spikeinExt option?

bam header

```bash

@hd VN:1.5 SO:coordinate
@sq SN:chr1 LN:195471971
@sq SN:chr2 LN:182113224
@sq SN:chr3 LN:160039680
@sq SN:chr4 LN:156508116
@sq SN:chr5 LN:151834684
@sq SN:chr6 LN:149736546
@sq SN:chr7 LN:145441459
@sq SN:chr8 LN:129401213
@sq SN:chr9 LN:124595110
@sq SN:chr10 LN:130694993
@sq SN:chr11 LN:122082543
@sq SN:chr12 LN:120129022
@sq SN:chr13 LN:120421639
@sq SN:chr14 LN:124902244
@sq SN:chr15 LN:104043685
@sq SN:chr16 LN:98207768
@sq SN:chr17 LN:94987271
@sq SN:chr18 LN:90702639
@sq SN:chr19 LN:61431566
@sq SN:chrX LN:171031299
@sq SN:chrY LN:91744698
@sq SN:chrM LN:16299
@sq SN:GL456210.1 LN:169725
@sq SN:GL456211.1 LN:241735
@sq SN:GL456212.1 LN:153618
@sq SN:GL456213.1 LN:39340
@sq SN:GL456216.1 LN:66673
@sq SN:GL456219.1 LN:175968
@sq SN:GL456221.1 LN:206961
@sq SN:GL456233.1 LN:336933
@sq SN:GL456239.1 LN:40056
@sq SN:GL456350.1 LN:227966
@sq SN:GL456354.1 LN:195993
@sq SN:GL456359.1 LN:22974
@sq SN:GL456360.1 LN:31704
@sq SN:GL456366.1 LN:47073
@sq SN:GL456367.1 LN:42057
@sq SN:GL456368.1 LN:20208
@sq SN:GL456370.1 LN:26764
@sq SN:GL456372.1 LN:28664
@sq SN:GL456378.1 LN:31602
@sq SN:GL456379.1 LN:72385
@sq SN:GL456381.1 LN:25871
@sq SN:GL456382.1 LN:23158
@sq SN:GL456383.1 LN:38659
@sq SN:GL456385.1 LN:35240
@sq SN:GL456387.1 LN:24685
@sq SN:GL456389.1 LN:28772
@sq SN:GL456390.1 LN:24668
@sq SN:GL456392.1 LN:23629
@sq SN:GL456393.1 LN:55711
@sq SN:GL456394.1 LN:24323
@sq SN:GL456396.1 LN:21240
@sq SN:JH584292.1 LN:14945
@sq SN:JH584293.1 LN:207968
@sq SN:JH584294.1 LN:191905
@sq SN:JH584295.1 LN:1976
@sq SN:JH584296.1 LN:199368
@sq SN:JH584297.1 LN:205776
@sq SN:JH584298.1 LN:184189
@sq SN:JH584299.1 LN:953012
@sq SN:JH584300.1 LN:182347
@sq SN:JH584301.1 LN:259875
@sq SN:JH584302.1 LN:155838
@sq SN:JH584303.1 LN:158099
@sq SN:JH584304.1 LN:114452
@sq SN:Chromosome LN:4686137
@rg ID:A006200317_201074_S18_L000 DS:A006200317_201074_S18_L000 PL:ILLUMINA SM:A006200317_201074_S18_L000
@pg ID:bowtie2 PN:bowtie2 CL:"mambaforge/envs/22c8c2155516caee04b33c92cdb28191/bin/bowtie2-align-s --wrapper basic-0 -X 1000 -x CUT-RUNTools-2.0/assemblies/BowtieIndex/genome --fr --rg-id A006200317_201074_S18_L000 --rg DS:A006200317_201074_S18_L000 --rg PL:ILLUMINA --rg SM:A006200317_201074_S18_L000 -p 8 -1 FASTQ_fastp/A006200317_201074_S18_L000_R1_001.fastq.gz -2 FASTQ_fastp/A006200317_201074_S18_L000_R2_001.fastq.gz" VN:2.4.5
@pg ID:samtools PN:samtools CL:samtools view -Sb - PP:bowtie2 VN:1.15.1
@pg ID:samtools.1 PN:samtools CL:samtools sort -m 2G -T /tmp//snakepipes.CrgbFQGyqz/A006200317_201074_S18_L000 -@ 2 -O bam - PP:samtools VN:1.15.1
@pg ID:sambamba CL:markdup -t 8 --sort-buffer-size=6000 --overflow-list-size 600000 --tmpdir /tmp//snakepipes.9Jiva0fp88 Bowtie2/A006200317_201074_S18_L000.sorted.bam Bowtie2/A006200317_201074_S18_L000.bam PP:samtools.1 VN:1.0
@pg ID:samtools.2 PN:samtools PP:sambamba VN:1.15.1 CL:samtools view -@ 2 -b -F 1024 -q 2 -o filtered_bam/A006200317_201074_S18_L000.filtered.tmp.bam Bowtie2/A006200317_201074_S18_L000.bam
@pg ID:samtools.3 PN:samtools PP:samtools.2 VN:1.15.1 CL:samtools view -h /mnt/c/AP01/bamSpikes/filtered_bam/A006200317_201074_S18_L000.filtered.bam
A00620:317:HCJLNDSX7:2:2426:19678:32095 163 chr1 3003373 42 101M = 3003505 233 TGGACTCGTGAGACAAGATGGCTCCTTCACCTGCTCTGGGGGTCAGAACCCTCCCAGGTGGCCACCTCTCCTGTGGCGGGGAAGGTACCTGAAAGTCTTCA FFFFFFFF:FFFFFFFF,FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFF AS:i:-15 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:41A34T14G9 YS:i:-5 YT:Z:CP RG:Z:A006200317_201074_S18_L000

</details>

Chromosome entries in the bam files.

```bash
for bam in /mnt/c/AP01/bamSpikes/filtered_bam/*bam; do echo $bam; samtools view $bam | grep Chromosome | wc -l; done
/mnt/c/AP01/bamSpikes/filtered_bam/A006200317_201074_S18_L000.filtered.bam
3354
/mnt/c/AP01/bamSpikes/filtered_bam/A006200317_201076_S19_L000.filtered.bam
2853
/mnt/c/AP01/bamSpikes/filtered_bam/A006200317_201078_S20_L000.filtered.bam
3826
/mnt/c/AP01/bamSpikes/filtered_bam/A006200317_201080_S21_L000.filtered.bam
1675
/mnt/c/AP01/bamSpikes/filtered_bam/A006200317_201082_S22_L000.filtered.bam
3535
/mnt/c/AP01/bamSpikes/filtered_bam/A006200317_201084_S23_L000.filtered.bam
1804

[katsikora]

Hi Sunta3iouxos,

your workflow in general looks good, i.e. createIndices -> DNA-mapping -> ChIPseq.

What does grep -c _spikein CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/genome_fasta/genome.fa return?
It looks like the _spikein postfix was not automatically appended by createIndices.

Best wishes,

Katarzyna

[sunta3iouxos]

0
but

grep -c Chromosome CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spik
es/genome_fasta/genome.fa
1

[katsikora]

What does the chromosome name in the bacterial fasta look like? Are there any spaces in it? This typically causes issues.

[sunta3iouxos]

 grep ">" CUT-RUNTools-2.0/assemblies/EB1/Sequence/WholeGenomeFasta/
genome.fa
>Chromosome

grep -c " " CUT-RUNTools-2.0/assemblies/EB1/Sequence/WholeGenomeFas
ta/genome.fa
0

[sunta3iouxos]

I added the

--spikeinExt Chromosome

and it does not complain. Is this correct then?

P.S. There are some other warnings, and messages that require new tickets.

[katsikora]

You mean you passed this to ChIP-seq? It might actually work.
Still, something went wrong in the createIndices instance. Have a look what the full chromosome name looks like, whether there are any characters in there that might bork up the renaming.

With snakePipes 2.7.2, I was able to create a hybrid genome with createIndices as:

createIndices -o $output/GRch38_Ecoli --tools bowtie2 --genomeURL /$genome/genome.fa --gtfURL $genes/genes.gtf --spikeinGenomeURL $genome/Ecoli_C3103_clean.fasta --userYAML GRCh38_g31_Ecoli

The chromosome name in the E.coli fasta was renamed from >CP053595.1 Escherichia coli strain T7Express_LysYIq chromosome, complete genome to >CP053595.1_spikein Escherichia coli strain T7Express_LysYIq chromosome, complete genome .

If you'd like to troubleshoot what happens with the chromosome names in the createIndices` worflow, you can pass `--snakemakeOptions ` --notemp ` , which would keep the temporary intermediate files.

Best wishes,

Katarzyna

[sunta3iouxos]

You mean you passed this to ChIP-seq? It might actually work.

Yes, using the

. Spikein chromosome extention can be specified with --spikeinExt.

[sunta3iouxos]

Still, something went wrong in the createIndices instance. Have a look what the full chromosome name looks like, whether there are any characters in there that might bork up the renaming

If you follow a bit the provided information, the _spikein identifier is there but only in the "/spikein_genes.gtf " but nowhere else (at least not in the readable files (so excluding the binary index bowtie2 files.

The genome.fa of the bacteria is as follows:

head CUT-RUNTools-2.0/assemblies/EB1/Sequence/WholeGenomeFasta/genome.fa
>Chromosome
AGCTTTTCATTCTGACTGCAACGGGCAATATGTCTCTGTGTGGATTAAAAAAAGAGTGTC
TGATAGCAGCTTCTGAACTGGTTACCTGCCGTGAGTAAATTAAAATTTTATTGACTTAGG
TCACTAAATACTTTAACCAATATAGGCATAGCGCACAGACAGATAAAAATTACAGAGTAC
ACAACATCCATGAAACGCATTAGCACCACCATTACCACCACCATCACCATTACCACAGGT
AACGGTGCGGGCTGACGCGTACAGGAAACACAGAAAAAAGCCCGCACCTGACAGTGCGGG
CTTTTTTTTTCGACCAAAGGTAACGAGGTAACAACCATGCGAGTGTTGAAGTTCGGCGGT
ACATCAGTGGCAAATGCAGAACGTTTTCTGCGTGTTGCCGATATTCTGGAAAGCAATGCC
AGGCAGGGGCAGGTGGCCACCGTCCTCTCTGCCCCCGCCAAAATCACCAACCACCTGGTG
GCGATGATTGAAAAAACCATTAGCGGCCAGGATGCTTTACCCAATATCAGCGATGCCGAA

I do not see any specific error.
The generated genome.fa in the spiked-in folder looks like:

grep ">" CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/genome_fasta/genome.fa
>chr1 1
>chr2 2
>chr3 3
>chr4 4
>chr5 5
>chr6 6
>chr7 7
>chr8 8
>chr9 9
>chr10 10
>chr11 11
>chr12 12
>chr13 13
>chr14 14
>chr15 15
>chr16 16
>chr17 17
>chr18 18
>chr19 19
>chrX X
>chrY Y
>chrM MT
>GL456210.1 GL456210.1
>GL456211.1 GL456211.1
>GL456212.1 GL456212.1
>GL456213.1 GL456213.1
>GL456216.1 GL456216.1
>GL456219.1 GL456219.1
>GL456221.1 GL456221.1
>GL456233.1 GL456233.1
>GL456239.1 GL456239.1
>GL456350.1 GL456350.1
>GL456354.1 GL456354.1
>GL456359.1 GL456359.1
>GL456360.1 GL456360.1
>GL456366.1 GL456366.1
>GL456367.1 GL456367.1
>GL456368.1 GL456368.1
>GL456370.1 GL456370.1
>GL456372.1 GL456372.1
>GL456378.1 GL456378.1
>GL456379.1 GL456379.1
>GL456381.1 GL456381.1
>GL456382.1 GL456382.1
>GL456383.1 GL456383.1
>GL456385.1 GL456385.1
>GL456387.1 GL456387.1
>GL456389.1 GL456389.1
>GL456390.1 GL456390.1
>GL456392.1 GL456392.1
>GL456393.1 GL456393.1
>GL456394.1 GL456394.1
>GL456396.1 GL456396.1
>JH584292.1 JH584292.1
>JH584293.1 JH584293.1
>JH584294.1 JH584294.1
>JH584295.1 JH584295.1
>JH584296.1 JH584296.1
>JH584297.1 JH584297.1
>JH584298.1 JH584298.1
>JH584299.1 JH584299.1
>JH584300.1 JH584300.1
>JH584301.1 JH584301.1
>JH584302.1 JH584302.1
>JH584303.1 JH584303.1
>JH584304.1 JH584304.1
>Chromosome

in the genome.fa.fai the correct size is reported:

grep Chromosome CUT-RUNTools-2.0/assemblies/mm10_gencodeM1
9_spikes/genome_fasta/genome.fa.fai
Chromosome      4686137 2776387558      60      61

INTERESTINGLY

grep Chromosome  /home/tgeorgom/CUT-RUNTools-2.0/assemblies/mm10_gencodeM19_spikes/annotation/spikein_genes.gtf | head -n 1
**Chromosome_spikein**      ena     CDS     190     252     .       +       0       exon_number "1"; gene_biotype "protein_coding"; gene_id "ECDH10B_0001"; gene_name "thrL"; gene_source "ena"; gene_version "1"; p_id "P2524"; protein_id "ACB01206"; protein_version "1"; transcript_biotype "protein_coding"; transcript_id "ACB01206"; transcript_name "thrL-1"; transcript_source "ena"; transcript_version "1"; tss_id "TSS3237";

[katsikora]

I can check if I can reproduce this issue here.

[sunta3iouxos]

Hi there,
any updates on this one? did you have time to check this?

[katsikora]

Hi,

could you send me the link to this bacterial genome fasta?

Best wishes,

Katarzyna