erro in step1 coassembly~

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Hello Professor, I have five samples here that have been individually spliced. I want to mix and splice them again, but I only have spliced data in FASTA format. I'm not sure if the 'extassembly' parameter is set incorrectly. Shotgun_samplefiles1.txt squeezemeta1-slurmtxt.txt

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/public/home/zhanglj/miniconda3/envs/squeezeMeta/SqueezeMeta/scripts/SqueezeMeta.pl -m coassembly -p shotgun1 -s Shotgun_samplefiles1.txt -f /public/home/zhanglj/Squeezemeta_20240108/assmblydata/ --euk -taxbinmode s+c -t 64

[jtamames]

Hello Looks good to me.. are you running in any error?

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你好， 我觉得不错..你运行时有什么错误吗？

Thank you for your response, Professor. Here is the error message I encountered. syslog.txt

step1:I have two sets of parallel samples. In each set, there are two files, such as FQ11 and FQ12 in the first set (FQ1 group). Initially, I merge the data from these two individual files within each set, and then I use the resulting combined data as input for further analysis.

step2: run pipline

[jtamames]

I don´t know if I understand correctly what you want to do. Do you want to analyze your five samples separately? If so, then specify mode sequential (-m sequential) and try again, it should work. Best, J

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Professor, i want to five smaples mix as one sample to analysis (coassembly)

[jtamames]

Then just run the coassembly, but don´t specify the extassembly in the samples file

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Thank you very much for your guidance. I understand now, and I will continue with my attempts accordingly.

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STEP10 -> 10.mapsamples.pl erro syslog.txt

Hello professor, I was able to run it normally under your guidance yesterday, but today I found an error in step 10 stopping;

[19 hours, 13 minutes, 34 seconds]: STEP10 -> 10.mapsamples.pl Creating Bowtie reference: python3 /mnt/hpc/home/zhanglj/miniconda3/envs/squeezeMeta/SqueezeMeta/bin/bowtie2/bowtie2-build --quiet /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/results/01.shotgun1.fasta /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/shotgun1.bowtie Getting raw reads for FQ1: cp /public/home/zhanglj/Squeezemeta_20240108/assmblydata//FQ1.fasta /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/temp/shotgun1.FQ1.current_1; Aligning with bowtie: /mnt/hpc/home/zhanglj/miniconda3/envs/squeezeMeta/SqueezeMeta/bin/bowtie2/bowtie2 -x /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/shotgun1.bowtie -f -U /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/temp/shotgun1.FQ1.current_1 --quiet -p 64 -S /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/bam/shotgun1.FQ1.sam --very-sensitive-local Stopping in STEP10 -> 10.mapsamples.pl. Program finished abnormally

[jtamames]

Try running this command and see if it works:

/mnt/hpc/home/zhanglj/miniconda3/envs/squeezeMeta/SqueezeMeta/bin/bowtie2/bowtie2 -x /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/shotgun1.bowtie -f /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/temp/shotgun1.FQ1.current_1 -U /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/temp/shotgun1.FQ1.current_1 --quiet -p 64 -S /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/bam/shotgun1.FQ1.sam --very-sensitive-local

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no results。

[jtamames]

The command is a single line. You typed it as several separated lines

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(squeezeMeta) [zhanglj@admin Squeezemeta_20240108]$ /mnt/hpc/home/zhanglj/miniconda3/envs/squeezeMeta/SqueezeMeta/bin/bowtie2/bowtie2 -x /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/shotgun1.bowtie -U /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/temp/shotgun1.FQ1.current_1 --quiet -p 64 -S /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/bam/shotgun1.FQ1.sam --very-sensitive-local

Error: reads file does not look like a FASTQ file terminate called after throwing an instance of 'int' (ERR): bowtie2-align died with signal 6 (ABRT) (core dumped)

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该命令是一行。您将其输入为几行分隔的行

Hello professor, it seems that there is a problem with my input file. Is it because I use the spliced (fasta file) as input? In this case, how should I modify it?

[jtamames]

This is because you are using a single unpaired fasta file, which is rather unusual. Try this:

/mnt/hpc/home/zhanglj/miniconda3/envs/squeezeMeta/SqueezeMeta/bin/bowtie2/bowtie2 -x /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/shotgun1.bowtie -f /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/temp/shotgun1.FQ1.current_1 --quiet -p 64 -S /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/bam/shotgun1.FQ1.sam --very-sensitive-local

in a single line, as above. This should produce a /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/bam/shotgun1.FQ1.sam file.

[Panda-smile]

这是因为您使用的是单个未配对的 fasta 文件，这是相当不寻常的。尝试这个：

/mnt/hpc/home/zhanglj/miniconda3/envs/squeezeMeta/SqueezeMeta/bin/bowtie2/bowtie2 -x /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/shotgun1.bowtie -f /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/temp/shotgun1.FQ1.current_1 --quiet -p 64 -S /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/bam/shotgun1.FQ1.sam --very-sensitive-local

在一行中，如上所述。这应该会生成一个/mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/bam/shotgun1.FQ1.sam文件。

Yes, it can run when I change the parameter to -f. How should I modify it in script.pl? (The reason is: there was insufficient memory when coassembly splicing the 100G original data; I could only splice a single piece first and then use it as input to continue the mixed assembly)

[Panda-smile]

meighit results is a （.fasta ）type, so i used the results as input.

[jtamames]

Ok, then do the following: 1) Locate where your SqueezeMeta is installed. You can know this using the command conda info --envs. Look for "SqueezeMeta". The listed path is where all the scripts from SqueezeMeta are stored. 2) Edit the script 10.mapsamples.pl in the scripts directory. Look for the following lines (lines 242-243 in the latest version)

if(-e "$tempdir/$par2name") { $command="$bowtie2_x_soft -x $bowtieref $formatoption -1 $tempdir/$par1name -2 $tempdir/$par2name --quiet -p $numthreads -S $samfile $mapping_options"; }
else { $command="$bowtie2_x_soft -x $bowtieref $formatoption -U $tempdir/$par1name  --quiet -p $numthreads -S $samfile $mapping_options"; } }

3) Modify these in this way:

if(-e "$tempdir/$par2name") { $command="$bowtie2_x_soft -x $bowtieref $formatoption -1 $tempdir/$par1name -2 $tempdir/$par2name --quiet -p $numthreads -S $samfile $mapping_options"; }
elsif($formatseq eq "fasta")  { $command="$bowtie2_x_soft -x $bowtieref $formatoption $tempdir/$par1name  --quiet -p $numthreads -S $samfile $mapping_options"; } 
else { $command="$bowtie2_x_soft -x $bowtieref $formatoption -U $tempdir/$par1name  --quiet -p $numthreads -S $samfile $mapping_options"; } }

4) Restart SqueezeMeta from step 10: /public/home/zhanglj/miniconda3/envs/squeezeMeta/SqueezeMeta/scripts/SqueezeMeta.pl -p shotgun1 --restart -step 10

This should do it

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Thank you very much for the professor's guidance. I have modified it and it is running now.

[jtamames]

Nice to hear. Closing issue

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I'm very sorry, Professor, it didn't work after re-running~ Is it because of memory? (Single node is 520G, I set it to 480G memory) Or is it because of other reasons?

squeezemeta1.txt syslog.txt 10.mapsamples.txt slurm-116892.txt

[jtamames]

According to the picture, I see that you didn´t do the changes that I suggested. Please follow the instructions above and try again.

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10.mapsamples.txt I just sent the wrong screenshot. Sorry.

[jtamames]

What happens if you run: /mnt/hpc/home/zhanglj/miniconda3/envs/squeezeMeta/SqueezeMeta/bin/bowtie2/bowtie2 -x /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/shotgun1.bowtie -f /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/temp/shotgun1.FQ1.current_1 -p 64 -S /mnt/hpc/home/zhanglj/Squeezemeta_20240108/shotgun1/data/bam/shotgun1.FQ1.sam --very-sensitive-local

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(base) [zhanglj@admin Squeezemeta_20240108]$ cat slurm-116895.out terminate called after throwing an instance of 'std::bad_alloc' what(): std::bad_alloc

slurm.out display as above；but jobs still running，

ending sliurm.out results : (base) [zhanglj@admin Squeezemeta_20240108]$ cat slurm-116897.out Error: Out of memory allocating 2147483648 __m128i's for DP matrix: 'std::bad_alloc' Tried to allocate DP matrix with 10498 columns, 30258 vectors per column, and and 4 vectors per cell terminate called after throwing an instance of 'std::exception' what(): std::exception (ERR): bowtie2-align died with signal 6 (ABRT) (core dumped)

Tried lowering the threads to 4 or 32 and still can't run（inital -t 64）