All frames are zero!

[yzhzu]

Hi, After update snippy, I used snippy again, the error happened.

the error information:

snpEff build -c reference/snpeff.config -dataDir . -gff3 ref

WARNING: All frames are zero! This seems rather odd, please check that 'frame' information in your 'genes' file is accurate.

bwa mem -Y -M -R '@RG\tID:New\tSM:New' -t 8 reference/ref.fa fake_reads.fq | samclip --max 10 --ref reference/ref.fa.fai | samtools

sort -n -l 0 -T /tmp --threads 3 -m 2000M | samtools fixmate -m --threads 3 - - | samtools sort -l 0 -T /tmp --threads 3 -m 2000M | sam tools markdup -T /tmp --threads 3 -r -s - - > d5BvsDSM.bam

[markdup] warning: unable to calculate estimated library size. Read pairs 0 should be greater than duplicate pairs 0, which should both be non zero.

can anyone help me deal with the error? Thanks yzhzhu

[Michael-Sikorski]

I have subscribed, as I have the same error message:

[bam_sort_core] merging from 0 files and 7 in-memory blocks... samtools sort: couldn't allocate memory for bam_mem [15:37:11] Error running command, check mysnps/snps.log

snps.log copied below: `

echo snippy 4.6.0

cd .../snippy

/usr/local/bin/snippy --cpus 16 --outdir mysnps --ref ref.gbk --R1 R1.fastq.gz --R2 R2.fastq.gz

samtools faidx reference/ref.fa

bwa index reference/ref.fa

[bwa_index] Pack FASTA... 0.04 sec [bwa_index] Construct BWT for the packed sequence... [bwa_index] 1.98 seconds elapse. [bwa_index] Update BWT... 0.03 sec [bwa_index] Pack forward-only FASTA... 0.03 sec [bwa_index] Construct SA from BWT and Occ... 0.67 sec [main] Version: 0.7.17-r1188 [main] CMD: bwa index reference/ref.fa [main] Real time: 3.120 sec; CPU: 2.765 sec

mkdir -p reference/genomes && cp -f reference/ref.fa reference/genomes/ref.fa

ln -sf reference/ref.fa .

ln -sf reference/ref.fa.fai .

mkdir -p reference/ref && gzip -c reference/ref.gff > reference/ref/genes.gff.gz

snpEff build -c reference/snpeff.config -dataDir . -gff3 ref

WARNING: All frames are zero! This seems rather odd, please check that 'frame' information in your 'genes' file is accurate.

bwa mem -Y -M -R '@RG\tID:mysnps\tSM:mysnps' -t 16 reference/ref.fa .../R1.fastq.gz ...R2.fastq.gz | samclip --max 10 --ref reference/ref.fa.fai | samtools sort -n -l 0 -T /tmp --threads 7 -m 1000M | samtools fixmate -m --threads 7 - - | samtools sort -l 0 -T /tmp --threads 7 -m 1000M | samtools markdup -T /tmp --threads 7 -r -s - - > snps.bam

[markdup] error reading header`

[sharbie88]

I also had a similar error as above:

snpEff build -c reference/snpeff.config -dataDir . -gff3 ref

WARNING: All frames are zero! This seems rather odd, please check that 'frame' information in your 'genes' file is accurate.

bwa mem -Y -M -R '@RG\tID:PC0073a-S0073\tSM:PC0073a-S0073' -t 8 reference/ref.fa subsampled.S0073_SS18M4947_R1.fastq subsampled.S0073_SS18M4947_R2.fastq | samclip --max 10 --ref reference/ref.fa.fai | samtools sort -n -l 0 -T /tmp --threads 3 -m 2000M | samtools fixmate -m --threads 3 - - | samtools sort -l 0 -T /tmp --threads 3 -m 2000M | samtools markdup -T /tmp --threads 3 -r -s - - > snps.bam

[markdup] error reading header`

Did you have any luck sorting it out?

[DavidL-H]

I have the same error when running: snippy --cpus 8 --outdir AcisSNPs --ref BW25113.fasta --R1 NG-30008_Acies_lib593532_8049_2_1.fastq --R2 NG-30008_Acies_lib593532_8049_2_2.fastq

Error message in snps.log:

echo snippy 4.6.0

cd /home/david/snippy results

/home/david/miniconda3/envs/snippy/bin/snippy --cpus 8 --outdir AcisSNPs --ref BW25113.fasta --R1 NG-30008_Acies_lib593532_8049_2_1.fastq --R2 NG-30008_Acies_lib593532_8049_2_2.fastq

samtools faidx reference/ref.fa

bwa index reference/ref.fa

[bwa_index] Pack FASTA... 0.03 sec [bwa_index] Construct BWT for the packed sequence... [bwa_index] 1.08 seconds elapse. [bwa_index] Update BWT... 0.02 sec [bwa_index] Pack forward-only FASTA... 0.02 sec [bwa_index] Construct SA from BWT and Occ... 0.37 sec [main] Version: 0.7.17-r1188 [main] CMD: bwa index reference/ref.fa [main] Real time: 1.551 sec; CPU: 1.516 sec

mkdir -p reference/genomes && cp -f reference/ref.fa reference/genomes/ref.fa

ln -sf reference/ref.fa .

ln -sf reference/ref.fa.fai .

mkdir -p reference/ref && gzip -c reference/ref.gff > reference/ref/genes.gff.gz

bwa mem -Y -M -R '@RG\tID:AcisSNPs\tSM:AcisSNPs' -t 8 reference/ref.fa /home/david/snippy results/NG-30008_Acies_lib593532_8049_2_1.fastq /home/david/snippy results/NG-30008_Acies_lib593532_8049_2_2.fastq | samclip --max 10 --ref reference/ref.fa.fai | samtools sort -n -l 0 -T /tmp --threads 3 -m 2000M | samtools fixmate -m --threads 3 - - | samtools sort -l 0 -T /tmp --threads 3 -m 2000M | samtools markdup -T /tmp --threads 3 -r -s - - > snps.bam

[markdup] error reading header

[ItxaSarko]

How did you manage to solve it? I have the same error:

echo snippy 4.6.0 cd /home/uoc/prueba /home/uoc/snippy/bin/snippy --cpus 2 --ref pseudomonas.fna --R1 1pio_R1.fastq.gz --R2 1pio_R2.fastq.gz --outdir /home/uoc/variant samtools faidx reference/ref.fa bwa index reference/ref.fa [bwa_index] Pack FASTA... 0.08 sec [bwa_index] Construct BWT for the packed sequence... [bwa_index] 1.79 seconds elapse. [bwa_index] Update BWT... 0.04 sec [bwa_index] Pack forward-only FASTA... 0.03 sec [bwa_index] Construct SA from BWT and Occ... 0.77 sec [main] Version: 0.7.17-r1188 [main] CMD: bwa index reference/ref.fa [main] Real time: 2.796 sec; CPU: 2.715 sec

mkdir -p reference/genomes && cp -f reference/ref.fa reference/genomes/ref.fa ln -sf reference/ref.fa . ln -sf reference/ref.fa.fai . mkdir -p reference/ref && gzip -c reference/ref.gff > reference/ref/genes.gff.gz bwa mem -Y -M -R '@rg\tID:variant\tSM:variant' -t 2 reference/ref.fa /home/uoc/prueba/1pio_R1.fastq.gz /home/uoc/prueba/1pio_R2.fastq.gz | samclip --max 10 --ref reference/ref.fa.fai | samtools sort -n -l 0 -T /tmp --threads 0 -m 8000M | samtools fixmate -m --threads 0 - - | samtools sort -l 0 -T /tmp --threads 0 -m 8000M | samtools markdup -T /tmp --threads 0 -r -s - - > snps.bam markdup: invalid option -- 'T'

Usage: samtools markdup

Option: -r Remove duplicate reads -l Max read length (default 300 bases) -s Report stats. --input-fmt-option OPT[=VAL] Specify a single input file format option in the form of OPTION or OPTION=VALUE -O, --output-fmt FORMAT[,OPT[=VAL]]... Specify output format (SAM, BAM, CRAM) --output-fmt-option OPT[=VAL] Specify a single output file format option in the form of OPTION or OPTION=VALUE --reference FILE Reference sequence FASTA FILE [null] -@, --threads INT Number of additional threads to use [0]

The input file must be coordinate sorted and must have gone through fixmates with the mate scoring option on.

Can you help me? I try `--cpus 1--ram 3 but nothing.

Thank you!!!