Problem with NextDenovo fungal assembly

Question

[naveedkhanchemist]

Hi guys, I am new in Genome assembly, I have fungus genome sequencing data from Oxford Nano Tech with 200 X coverage. I used NextDenovo for assembly. I am using my personal system with 32 GB RAM. The parameter file I set is like this,

***[General]
job_type = local
job_prefix = nextDenovo
task = all # 'all', 'correct', 'assemble'
rewrite = yes # yes/no
deltmp = yes
rerun = 3
parallel_jobs = 2
input_type = raw
read_type = ont
input_fofn = ../input.fofn
workdir = ../assembly_number_3
[correct_option]
read_cutoff = 1k
genome_size = 45mb
pa_correction = 3
sort_options = -m 25g -t 5
minimap2_options_raw =  -t 5
correction_options = -p 8
[assemble_option]
minimap2_options_cns =  -t 5
nextgraph_options = -a 1***


I got an error like this,
[396803 CRITICAL] 2023-01-23 01:54:27 Command '/bin/sh /home/navi/magnaporthe-KI215/assembly_number_3/01.raw_align/04.sort_align.sh.work/sort_align2/nextDenovo.sh > /home/navi/magnaporthe-KI215/assembly_number_3/01.raw_align/04.sort_align.sh.work/sort_align2/nextDenovo.sh.o 2> /home/navi/magnaporthe-KI215/assembly_number_3/01.raw_align/04.sort_align.sh.work/sort_align2/nextDenovo.sh.e' returned non-zero exit status 137, error info: 


They asked me to check,
 sort_align failed: please check the following logs:
[393081 ERROR] 2023-01-23 01:55:20 /home/navi/magnaporthe-KI215/assembly_number_3/01.raw_align/04.sort_align.sh.work/sort_align1/nextDenovo.sh.e


This is the log. sh.e file:
hostname
+ hostname
cd /home/navi/magnaporthe-KI215/assembly_number_3/01.raw_align/03.raw_align.sh.work/raw_align4
+ cd /home/navi/magnaporthe-KI215/assembly_number_3/01.raw_align/03.raw_align.sh.work/raw_align4
time /home/navi/magnaporthe-KI215/NextDenovo/bin/minimap2-nd --step 1 -I 3G --dual=yes -t 5 -x ava-ont /home/navi/magnaporthe-KI215/NextDenovo/../assembly_number_3/01.raw_align/input.seed.001.2bit /home/navi/magnaporthe-KI215/NextDenovo/../assembly_number_3/01.raw_align/input.seed.002.2bit -o input.seed.001.2bit.3.ovl;ln -sf input.seed.001.2bit.3.ovl input.seed.002.2bit.3.ovl;
+ time /home/navi/magnaporthe-KI215/NextDenovo/bin/minimap2-nd --step 1 -I 3G --dual=yes -t 5 -x ava-ont /home/navi/magnaporthe-KI215/NextDenovo/../assembly_number_3/01.raw_align/input.seed.001.2bit /home/navi/magnaporthe-KI215/NextDenovo/../assembly_number_3/01.raw_align/input.seed.002.2bit -o input.seed.001.2bit.3.ovl
[M::mm_idx_gen::9.332*1.56] collected minimizers
[M::mm_idx_gen::11.732*2.25] sorted minimizers
[M::main::11.732*2.25] loaded/built the index for 17850 target sequence(s)
[M::mm_mapopt_update::12.616*2.17] mid_occ = 439
[M::mm_idx_stat] kmer size: 15; skip: 5; is_hpc: 0; #seq: 17850
[M::mm_idx_stat::13.227*2.11] distinct minimizers: 63051508 (63.44% are singletons); average occurrences: 3.650; average spacing: 2.932
[M::worker_pipeline::150.032*4.74] mapped 13209 sequences
[M::worker_pipeline::200.613*4.79] mapped 4641 sequences
[M::main] Version: 2.17-r941
[M::main] CMD: /home/navi/magnaporthe-KI215/NextDenovo/bin/minimap2-nd --step 1 -I 3G --dual=yes -t 5 -x ava-ont -o input.seed.001.2bit.3.ovl /home/navi/magnaporthe-KI215/NextDenovo/../assembly_number_3/01.raw_align/input.seed.001.2bit /home/navi/magnaporthe-KI215/NextDenovo/../assembly_number_3/01.raw_align/input.seed.002.2bit
[M::main] Real time: 200.725 sec; CPU: 960.890 sec; Peak RSS: 7.012 GB
956.25user 4.92system 3:21.05elapsed 478%CPU (0avgtext+0avgdata 7353052maxresident)k
330224inputs+311920outputs (0major+3705846minor)pagefaults 0swaps
+ ln -sf input.seed.001.2bit.3.ovl input.seed.002.2bit.3.ovl
touch /home/navi/magnaporthe-KI215/assembly_number_3/01.raw_align/03.raw_align.sh.work/raw_align4/nextDenovo.sh.done
+ touch /home/navi/magnaporthe-KI215/assembly_number_3/01.raw_align/03.raw_align.sh.work/raw_align4/nextDenovo.sh.done

I am confused about how to sort out this problem.... it seems like it is a memory issue..... any help in this regard is highly appreciated.

Answer 1

[colindaven]

• 200X coverage is too much.
• Reduce to eg 50X by using eg head -n 200000 x.fastq > x_small.fastq or similar (adjust the number to get roughly 50X)
• also try the assembler flye, it's great and far more widely used than nextdenovo
• 32GB is very low for assembly
• The shasta assembler is also great, but likely needs more RAM than you have

[samuel.a.odonnell]

I would just add to perhaps take the longest reads and removing very short ones while downsampling. A quick, easy tool for that is filtlong. Particularly when using flye, I find that removing short reads (>5kb) helps a lot with assembly.

[colindaven]

Yes, good point.

[tnyakauru]

Hi all

I am new to this. I am trying to install NextDenovo on ubuntu but I just cant get it to work. I am kindly asking if there is anyone who can help me on one on one.

Thank you