merging sequence highQuality-nanopore_mk1c.fastq.gz does not exist or size zero and Assembly with flye failed

[NurislamSheih]

Dear @alekseyzimin, I'm trying to assembly insect genome using trimmed illumina and trimmed nanopore data. I got the following errors in bold. However, the assembly in flye folder is succeeded, and there are no errors in the logs, at least I did not see. Сan there be any suggestions about nature of the problem? Thank you in advance

[Sun 20 Mar 2022 04:16:35 PM UTC] Processing pe library reads [Sun 20 Mar 2022 04:19:51 PM UTC] Average PE read length 229 [Sun 20 Mar 2022 04:19:51 PM UTC] Using kmer size of 83 for the graph [Sun 20 Mar 2022 04:19:51 PM UTC] MIN_Q_CHAR: 33 [Sun 20 Mar 2022 04:19:51 PM UTC] Creating mer database for Quorum [Sun 20 Mar 2022 04:24:23 PM UTC] Error correct PE [Sun 20 Mar 2022 04:37:39 PM UTC] Estimating genome size [Sun 20 Mar 2022 04:43:29 PM UTC] Estimated genome size: 234049591 [Sun 20 Mar 2022 04:43:29 PM UTC] Creating k-unitigs with k=83 [Sun 20 Mar 2022 05:16:18 PM UTC] Computing super reads from PE [Sun 20 Mar 2022 05:45:01 PM UTC] Using Flye from /home/nurislam/bioinfotools/MaSuRCA-4.0.8/bin/../Flye/bin [Sun 20 Mar 2022 05:45:01 PM UTC] Running mega-reads correction/assembly [Sun 20 Mar 2022 05:45:01 PM UTC] Using mer size 17 for mapping, B=15, d=0.02 [Sun 20 Mar 2022 05:45:01 PM UTC] Estimated Genome Size 234049591 [Sun 20 Mar 2022 05:45:01 PM UTC] Estimated Ploidy 1 [Sun 20 Mar 2022 05:45:01 PM UTC] Using 70 threads [Sun 20 Mar 2022 05:45:01 PM UTC] Output prefix mr.83.17.15.0.02 [Sun 20 Mar 2022 05:45:01 PM UTC] Creating k-unitigs for k=19 [Sun 20 Mar 2022 05:52:07 PM UTC] Pre-correcting long reads [Sun 20 Mar 2022 06:21:08 PM UTC] Pre-corrected reads are in longest_reads.25x.fa [Sun 20 Mar 2022 06:21:13 PM UTC] Computing mega-reads [Sun 20 Mar 2022 06:21:13 PM UTC] Running locally in 1 batch [Mon 21 Mar 2022 02:21:40 AM UTC] Refining alignments [Mon 21 Mar 2022 02:51:50 AM UTC] Computing allowed merges [Mon 21 Mar 2022 02:52:32 AM UTC] Joining [Mon 21 Mar 2022 02:59:32 AM UTC] Gap consensus [Mon 21 Mar 2022 03:04:15 AM UTC] Running assembly with Flye [Mon 21 Mar 2022 03:46:08 AM UTC] merging sequence highQuality-nanopore_mk1c.fastq.gz does not exist or size zero [Mon 21 Mar 2022 03:46:08 AM UTC] Assembly with flye failed, see files under flye/

[doenjon]

Hi,

I experienced the same "merging sequence X does not exist or size zero", despite an apparently successful flye assembly. It appears that my flye assembly is missing the assembly.scaffolds.fasta file that the assembly.sh script search for (even though it has a valid assembly.fasta file). The issue was caused by a conflicting version of flye in my path that doesn't produce a scaffolded assembly by default. Installing masurca in its own conda environment fixed the issue.

[alekseyzimin]

Hello, This error implies that post-processing of the flye assembly did not run. As a quick fix you can use the assembly.fasta under flye.mr...... folder. Please make sure you specify the full absolute path to the nanopore reads file in the masurca config file. Aleksey

On Tue, Jun 7, 2022 at 1:57 PM doenjon @.***> wrote:

Hi,

I experienced the same "merging sequence X does not exist or size zero", despite an apparently successful flye assembly. It appears that my flye assembly is missing the assembly.scaffolds.fasta file that the assembly.sh script search for (even though it has a valid assembly.fasta file), perhaps because my assembly did not result in any scaffolds.

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