5-untip/untip.sh run for a very long time?

[linyuiz]

Dear developer, hello, I have some questions I'd like to ask!
I am currently using verkko to assemble a homologous hexaploid using only HiFi data. The verkko strategy combining HiFi and ONT has already completed its run much earlier. But my HIFI-only strategy never seems to finish running.
Why does the script 5-untip/untip.sh take such a long time to run? It's been stuck at the following point for 3-4 days now:

Combine mappings
Combine edges
Find lengths
Fix coverage
Pop bubbles based on coverage

Is my genome too fragmented? The file nodecov_hifi_fix.csv has a lot of lines. What should I do to complete untip.sh faster?

cat nodecov_hifi_fix.csv|wc -l
150821

5-untip/untip.sh:

#!/bin/sh
set -e


 /home//anaconda3/envs/verkko/lib/verkko/scripts/untip_relative.py 15000 5000 0.1 <../2-processGraph/unitig-unrolled-hifi-resolved.gfa > connected-tip.gfa

 /home//anaconda3/envs/verkko/lib/verkko/scripts/unitigify.py utig3a- unitig-mapping-3a.txt \
 < connected-tip.gfa \
  > unitig-connected-tip.gfa

echo Combine mappings
cat ../2-processGraph/unitig-mapping-1.txt ../2-processGraph/haplofix-mapping.txt ../2-processGraph/unroll_mapping_1.txt  unitig-mapping-3a.txt \
> combined-nodemap-1.txt

echo Combine edges
cat ../1-buildGraph/hifi-resolved.gfa ../2-processGraph/gapped-once-hifi-resolved.gfa ../2-processGraph/gapped-twice-hifi-resolved.gfa ../2-processGraph/gapped-hifi-resolved.gfa ../2-processGraph/fixed-hifi-resolved.gfa ../2-processGraph/unrolled-hifi-resolved.gfa ../2-processGraph/unitig-unrolled-hifi-resolved.gfa  connected-tip.gfaunitig-connected-tip.gfa \
| grep '^L' \
> combined-edges-1.gfa


# this used to have grep -v \*
#
echo Find lengths
cat ../1-buildGraph/hifi-resolved.gfa ../2-processGraph/gapped-once-hifi-resolved.gfa ../2-processGraph/gapped-twice-hifi-resolved.gfa ../2-processGraph/gapped-hifi-resolved.gfa ../2-processGraph/fixed-hifi-resolved.gfa ../2-processGraph/unrolled-hifi-resolved.gfa ../2-processGraph/unitig-unrolled-hifi-resolved.gfa  connected-tip.gfaunitig-connected-tip.gfa \
| awk 'BEGIN \
       { \
          FS="[ \t]+"; OFS="\t"; \
       } \
       $1 == "S" \
       { \
           print $2, length($3); \
       }' \
> nodelens-1.txt

echo Fix coverage
 /home//anaconda3/envs/verkko/lib/verkko/scripts/get_original_coverage.py \
  unitig-connected-tip.gfa \
  combined-nodemap-1.txt \
  combined-edges-1.gfa \
  nodelens-1.txt \
  ../1-buildGraph/hifi_nodecov.csv \
> ./nodecov_hifi_fix.csv

echo Pop bubbles based on coverage
 /home//anaconda3/envs/verkko/lib/verkko/scripts/pop_bubbles_coverage_based.py \
  unitig-connected-tip.gfa \
  ./nodecov_hifi_fix.csv False \
> popped-connected-tip.gfa \
2> popinfo_round1.err

# consider doing this in a loop to avoid code redundancy
echo Unroll simple loops
 /home//anaconda3/envs/verkko/lib/verkko/scripts/unroll_simple_loops.py popped-connected-tip.gfa ./nodecov_hifi_fix.csv > unrolled-popped-connected-tip.gfa 2>> unitig-mapping-3a.txt

echo Unitigify 3
 /home//anaconda3/envs/verkko/lib/verkko/scripts/unitigify.py utig3b- unitig-mapping-3b.txt \
< unrolled-popped-connected-tip.gfa \
> unitig-unrolled-popped-connected-tip.gfa

echo Combine mappings
cat ../2-processGraph/unitig-mapping-1.txt ../2-processGraph/haplofix-mapping.txt ../2-processGraph/unroll_mapping_1.txt  unitig-mapping-3a.txt unitig-mapping-3b.txt \
> combined-nodemap-1.txt

echo Combine edges
cat ../1-buildGraph/hifi-resolved.gfa ../2-processGraph/gapped-once-hifi-resolved.gfa ../2-processGraph/gapped-twice-hifi-resolved.gfa ../2-processGraph/gapped-hifi-resolved.gfa ../2-processGraph/fixed-hifi-resolved.gfa ../2-processGraph/unrolled-hifi-resolved.gfa ../2-processGraph/unitig-unrolled-hifi-resolved.gfa  connected-tip.gfaunitig-connected-tip.gfa popped-connected-tip.gfa unrolled-popped-connected-tip.gfaunitig-unrolled-popped-connected-tip.gfa \
| grep '^L' \
> combined-edges-1.gfa


# this used to have grep -v \*
#
echo Find lengths
cat ../1-buildGraph/hifi-resolved.gfa ../2-processGraph/gapped-once-hifi-resolved.gfa ../2-processGraph/gapped-twice-hifi-resolved.gfa ../2-processGraph/gapped-hifi-resolved.gfa ../2-processGraph/fixed-hifi-resolved.gfa ../2-processGraph/unrolled-hifi-resolved.gfa ../2-processGraph/unitig-unrolled-hifi-resolved.gfa  connected-tip.gfaunitig-connected-tip.gfa popped-connected-tip.gfa unrolled-popped-connected-tip.gfaunitig-unrolled-popped-connected-tip.gfa \
| awk 'BEGIN \
       { \
          FS="[ \t]+"; OFS="\t"; \
       } \
       $1 == "S" \
       { \
           print $2, length($3); \
       }' \
> nodelens-1.txt

echo Fix coverage
 /home//anaconda3/envs/verkko/lib/verkko/scripts/get_original_coverage.py \
  ../5-untip/unitig-unrolled-popped-connected-tip.gfa \
  combined-nodemap-1.txt \
  combined-edges-1.gfa \
  nodelens-1.txt \
  ../1-buildGraph/hifi_nodecov.csv \
>> ./nodecov_hifi_fix.csv

echo Pop bubbles based on coverage round 2
 /home//anaconda3/envs/verkko/lib/verkko/scripts/pop_bubbles_coverage_based.py \
  unitig-unrolled-popped-connected-tip.gfa \
  ./nodecov_hifi_fix.csv False \
  > popped-unitig-unrolled-popped-connected-tip.gfa \
  2> popinfo_round2.err

echo Unroll simple loops round 2
 /home//anaconda3/envs/verkko/lib/verkko/scripts/unroll_simple_loops.py popped-unitig-unrolled-popped-connected-tip.gfa ./nodecov_hifi_fix.csv > unrolled-popped-unitig-unrolled-popped-connected-tip.gfa 2>> unitig-mapping-3b.txt

echo Unitigify 4
 /home//anaconda3/envs/verkko/lib/verkko/scripts/unitigify.py utig4- unitig-mapping-4.txt < unrolled-popped-unitig-unrolled-popped-connected-tip.gfa > ../5-untip/unitig-unrolled-unitig-unrolled-popped-unitig-normal-connected-tip.gfa

# disable until we support jump links
#
# > pregap-popped-unitig-normal-connected-tip.gfa
#
#echo Add homozygous node scaffold edges
#cat combined-nodemap-1.txt unitig-mapping-3a.txt unitig-mapping-3b.txt unitig-mapping-4.txt > combined-nodemap-2.txt
# /home//anaconda3/envs/verkko/lib/verkko/scripts/add_hom_node_scaffold_edges.py pregap-popped-unitig-normal-connected-tip.gfa combined-nodemap-2.txt homgap > ../5-untip/unitig-unrolled-unitig-unrolled-popped-unitig-normal-connected-tip.gfa


echo Generate Coverage

#
#   Generates files used to compute coverage tables.  It combines all the
#   various bits and pieces of the assembly into three outputs.  Which files
#   are combined depends on which stage we're at in the assembly process
#   (and if ONT data is included); getAllMappings() and getAllGraphs()
#   figures this out.
#      5-untip/combined-edges-final.gfa     #  Edges in the graphs
#      5-untip/nodelens-final.txt           #  Sequence lines that have actual sequence
#      5-untip/combined-nodemap-final.txt   #  Maps from nodes to components
#
#   Then computes coverage of either HiFi or ONT reads for unitigs at various stages.
#     hifi2cov = '2-processGraph/unitig-unrolled-hifi-resolved.hifi-coverage.csv',
#     ont2cov  = '2-processGraph/unitig-unrolled-hifi-resolved.ont-coverage.csv'   if config['withONT'] == "True" else rules.emptyfile.output,
#     hifi4cov = '4-processONT/unitig-unrolled-ont-resolved.hifi-coverage.csv'   if config['withONT'] == "True" else rules.emptyfile.output,
#     ont4cov  = '4-processONT/unitig-unrolled-ont-resolved.ont-coverage.csv'   if config['withONT'] == "True" else rules.emptyfile.output,
#     hifi5cov = '5-untip/unitig-unrolled-unitig-unrolled-popped-unitig-normal-connected-tip.hifi-coverage.csv',
#     ont5cov  = '5-untip/unitig-unrolled-unitig-unrolled-popped-unitig-normal-connected-tip.ont-coverage.csv'  if config['withONT'] == "True" else rules.emptyfile.output,

cat ../2-processGraph/unitig-mapping-1.txt ../2-processGraph/haplofix-mapping.txt ../2-processGraph/unroll_mapping_1.txt ../5-untip/unitig-mapping-3a.txt ../5-untip/unitig-mapping-3b.txt ../5-untip/unitig-mapping-4.txt  \
> ./combined-nodemap-final.txt

cat ../1-buildGraph/hifi-resolved.gfa ../2-processGraph/gapped-once-hifi-resolved.gfa ../2-processGraph/gapped-twice-hifi-resolved.gfa ../2-processGraph/gapped-hifi-resolved.gfa ../2-processGraph/fixed-hifi-resolved.gfa ../2-processGraph/unrolled-hifi-resolved.gfa ../2-processGraph/unitig-unrolled-hifi-resolved.gfa ../5-untip/connected-tip.gfa ../5-untip/unitig-connected-tip.gfa ../5-untip/popped-connected-tip.gfa ../5-untip/unrolled-popped-connected-tip.gfa ../5-untip/unitig-unrolled-popped-connected-tip.gfa ../5-untip/popped-unitig-unrolled-popped-connected-tip.gfa ../5-untip/unrolled-popped-unitig-unrolled-popped-connected-tip.gfa ../5-untip/unitig-unrolled-unitig-unrolled-popped-unitig-normal-connected-tip.gfa  \
| grep '^L' \
> ./combined-edges-final.gfa

cat ../1-buildGraph/hifi-resolved.gfa ../2-processGraph/gapped-once-hifi-resolved.gfa ../2-processGraph/gapped-twice-hifi-resolved.gfa ../2-processGraph/gapped-hifi-resolved.gfa ../2-processGraph/fixed-hifi-resolved.gfa ../2-processGraph/unrolled-hifi-resolved.gfa ../2-processGraph/unitig-unrolled-hifi-resolved.gfa ../5-untip/connected-tip.gfa ../5-untip/unitig-connected-tip.gfa ../5-untip/popped-connected-tip.gfa ../5-untip/unrolled-popped-connected-tip.gfa ../5-untip/unitig-unrolled-popped-connected-tip.gfa ../5-untip/popped-unitig-unrolled-popped-connected-tip.gfa ../5-untip/unrolled-popped-unitig-unrolled-popped-connected-tip.gfa ../5-untip/unitig-unrolled-unitig-unrolled-popped-unitig-normal-connected-tip.gfa  \
| awk 'BEGIN \
       { \
          FS="[ \t]+"; OFS="\t"; \
       } \
       ($1 == "S") && ($3 != "*") \
       { \
          print $2, length($3); \
       }' \
> ./nodelens-final.txt

for gg in 2-processGraph/unitig-unrolled-hifi-resolved \
          4-processONT/unitig-unrolled-ont-resolved \
          5-untip/unitig-unrolled-unitig-unrolled-popped-unitig-normal-connected-tip ; do
  if [ -e ../$gg.gfa ] ; then
     /home//anaconda3/envs/verkko/lib/verkko/scripts/get_original_coverage.py ../$gg.gfa ./combined-nodemap-final.txt ./combined-edges-final.gfa ./nodelens-final.txt ../1-buildGraph/hifi_nodecov.csv > ../$gg.hifi-coverage.csv
  fi
  if [ -e ../$gg.gfa ] ; then
     /home//anaconda3/envs/verkko/lib/verkko/scripts/get_original_coverage.py ../$gg.gfa ./combined-nodemap-final.txt ./combined-edges-final.gfa ./nodelens-final.txt ../emptyfile  > ../$gg.ont-coverage.csv
  fi
done

[skoren]

I suspect this is because your graph is very fragmented with only HiFi data. The step that's taking a while partitions the graph into connected components and does some other expensive graph analysis. I wouldn't bother finishing the run, you already have a HiFi-only graph from your combined HiFi + ONT run in the 2-processGraph folder. It wouldn't have the bubbles removed but would still give you an idea of what the HiFi assembly looks like.

[linyuiz]

Thank you for your reply. I do have a contig genome with HIFI+ONT, but I still want a version with the HIFI-Only strategy. If it's really not possible to change its operation by adjusting parameters, it seems I'll have to give up.

[skoren]

It's possible we could modify the script to not perform some simplifications on very large graphs or try to make it more efficient but given that verkko is primarily designed for HiFi + ONT it's not a high priority.

My point was you already have the hifi-only strategy assembly as subset of the HiFi+ONT assembly. The HiFi-only graph is in the 2-processGraph/*hifi-resolved.gfa file while the addition of ONT is in 4-processONT/*ont-resolved.gfa. These are in homopolymers-compressed space but you can still compare them in terms of continuity and number of nodes/edges to get an idea of the improvement from ONT integration.