Assembly correction tools: Polca, Pilon, and Medaka

1️⃣ Polca – A lightweight polishing tool from MaSuRCA that corrects small sequencing errors using short-read data. It efficiently fixes substitutions and small INDELs but is not ideal for large structural variations.

    # Under the env (nextclade)
    mamba install -c bioconda -c conda-forge masurca

    #-- VZV_20S.assembly3-modify.fasta --
    (nextclade) polca.sh -a ../viralngs/tmp/02_assembly/VZV_20S.assembly3-modify.fasta -r "VZV_20S_trimmed_P_1.fastq VZV_20S_trimmed_P_2.fastq" -t 40 -m 10G
    #3
    (nextclade) polca.sh -a VZV_20S.assembly3-modify.fasta.PolcaCorrected.fa -r "VZV_20S_trimmed_P_1.fastq VZV_20S_trimmed_P_2.fastq" -t 40 -m 10G
    #0

2️⃣ Pilon – A more comprehensive short-read polishing tool that corrects SNPs, small INDELs, and some structural misassemblies. It works best with high-coverage Illumina reads and can iteratively improve assembly accuracy.

    bwa index PCC1_VZV_20_2.assembly3-modify.fasta.PolcaCorrected.fa
    bwa mem -t 40 PCC1_VZV_20_2.assembly3-modify.fasta.PolcaCorrected.fa \
        PCC1_VZV_20_2_trimmed_P_1.fastq \
        PCC1_VZV_20_2_trimmed_P_2.fastq \
        > aln.sam
    samtools view -bS aln.sam > aln.bam
    samtools sort aln.bam aln.sorted
    samtools index aln.sorted.bam
    (nextclade) pilon --genome PCC1_VZV_20_2.assembly3-modify.fasta.PolcaCorrected.fa \
        --bam aln.sorted.bam --output polished --threads 80 --changes --fix indels

3️⃣ Medaka – A polishing tool specifically designed for Nanopore sequencing data. It uses a neural network to refine base calls and correct systematic errors in long-read assemblies.

1. Quality check using QUAST

   #mamba install -c bioconda quast
   #quast polished.fasta -r reference.fasta -o quast_output
   

2. Correcting assembly for Huang_Human_herpesvirus_3

   ./VZV_20S.fasta
   ./VZV_20c.fasta
   ./PCC1_VZV_20_1.fasta
   ./PCC1_VZV_20_2.fasta
   ./PCC1_VZV_20_5.fasta
   
   ./VZV_60S.fasta
   ./VZV_60c.fasta
   ./PCC1_VZV_60_1.fasta
   ./PCC1_VZV_60_4.fasta
   ./PCC1_VZV_60_6.fasta
   
   #find . -nma "*.assembly1-spades.fasta" | wc -l
   #find . -name "*.assembly2-gapfilled.fasta" | wc -l
   #find . -name "*.assembly3-modify.fasta" | wc -l
   #find . -name "*.assembly4-refined.fasta" | wc -l
   
   # Under the env (nextclade) and directory ~/DATA/Data_Huang_Human_herpesvirus_3/trimmed
   mamba install -c bioconda -c conda-forge masurca
   
   #-- VZV_20S.assembly3-modify.fasta --
   (nextclade) polca.sh -a ../viralngs/tmp/02_assembly/VZV_20S.assembly3-modify.fasta -r "VZV_20S_trimmed_P_1.fastq VZV_20S_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 3
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124870
   #Consensus Quality Before Polishing: 99.9976
   #Consensus QV Before Polishing: 46.19
   (nextclade) polca.sh -a VZV_20S.assembly3-modify.fasta.PolcaCorrected.fa -r "VZV_20S_trimmed_P_1.fastq VZV_20S_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124869
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   
   #-- VZV_20c.assembly3-modify.fasta --
   polca.sh -a ../viralngs/tmp/02_assembly/VZV_20c.assembly3-modify.fasta -r "VZV_20c_trimmed_P_1.fastq VZV_20c_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 2
   #Insertion/Deletion Errors Found: 2
   #Assembly Size: 124872
   #Consensus Quality Before Polishing: 99.9968
   #Consensus QV Before Polishing: 44.94
   polca.sh -a VZV_20c.assembly3-modify.fasta.PolcaCorrected.fa -r "VZV_20c_trimmed_P_1.fastq VZV_20c_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124873
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   
   #-- PCC1_VZV_20_1.assembly3-modify.fasta --
   polca.sh -a ../viralngs/tmp/02_assembly/PCC1_VZV_20_1.assembly3-modify.fasta -r "PCC1_VZV_20_1_trimmed_P_1.fastq PCC1_VZV_20_1_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 1
   #Insertion/Deletion Errors Found: 1
   #Assembly Size: 124873
   #Consensus Quality Before Polishing: 99.9984
   #Consensus QV Before Polishing: 47.95
   polca.sh -a PCC1_VZV_20_1.assembly3-modify.fasta.PolcaCorrected.fa -r "PCC1_VZV_20_1_trimmed_P_1.fastq PCC1_VZV_20_1_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 1
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124873
   #Consensus Quality Before Polishing: 99.9992
   #Consensus QV Before Polishing: 50.96
   polca.sh -a PCC1_VZV_20_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa -r "PCC1_VZV_20_1_trimmed_P_1.fastq PCC1_VZV_20_1_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124873
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   
   #-- PCC1_VZV_20_2.assembly3-modify.fasta --
   polca.sh -a ../viralngs/tmp/02_assembly/PCC1_VZV_20_2.assembly2-gapfilled.fasta -r "PCC1_VZV_20_2_trimmed_P_1.fastq PCC1_VZV_20_2_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 1
   #Insertion/Deletion Errors Found: 1
   #Assembly Size: 124866
   #Consensus Quality Before Polishing: 99.9984
   #Consensus QV Before Polishing: 47.95
   polca.sh -a PCC1_VZV_20_2.assembly2-gapfilled.fasta.PolcaCorrected.fa -r "PCC1_VZV_20_2_trimmed_P_1.fastq PCC1_VZV_20_2_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124867
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   
   #-- PCC1_VZV_20_5.assembly3-modify.fasta --
   polca.sh -a ../viralngs/tmp/02_assembly/PCC1_VZV_20_5.assembly3-modify.fasta -r "PCC1_VZV_20_5_trimmed_P_1.fastq PCC1_VZV_20_5_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 1
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124873
   #Consensus Quality Before Polishing: 99.9992
   #Consensus QV Before Polishing: 50.96
   polca.sh -a PCC1_VZV_20_5.assembly3-modify.fasta.PolcaCorrected.fa -r "PCC1_VZV_20_5_trimmed_P_1.fastq PCC1_VZV_20_5_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124874
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   
   #-- VZV_60S.assembly3-modify.fasta --
   polca.sh -a ../viralngs/tmp/02_assembly/VZV_60S.assembly3-modify.fasta -r "VZV_60S_trimmed_P_1.fastq VZV_60S_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 2
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124873
   #Consensus Quality Before Polishing: 99.9984
   #Consensus QV Before Polishing: 47.95
   polca.sh -a VZV_60S.assembly3-modify.fasta.PolcaCorrected.fa -r "VZV_60S_trimmed_P_1.fastq VZV_60S_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 1
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124870
   #Consensus Quality Before Polishing: 99.9992
   #Consensus QV Before Polishing: 50.96
   polca.sh -a VZV_60S.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa -r "VZV_60S_trimmed_P_1.fastq VZV_60S_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124870
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   
   #-- VZV_60c.assembly2-gapfilled.fasta --
   polca.sh -a ../viralngs/tmp/02_assembly/VZV_60c.assembly2-gapfilled.fasta -r "VZV_60c_trimmed_P_1.fastq VZV_60c_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 1
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 119660
   #Consensus Quality Before Polishing: 99.9992
   #Consensus QV Before Polishing: 50.78
   polca.sh -a VZV_60c.assembly2-gapfilled.fasta.PolcaCorrected.fa -r "VZV_60c_trimmed_P_1.fastq VZV_60c_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 119660
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   
   #-- PCC1_VZV_60_1.assembly3-modify.fasta --
   polca.sh -a ../viralngs/tmp/02_assembly/PCC1_VZV_60_1.assembly3-modify.fasta -r "PCC1_VZV_60_1_trimmed_P_1.fastq PCC1_VZV_60_1_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124843
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   polca.sh -a PCC1_VZV_60_1.assembly3-modify.fasta.PolcaCorrected.fa -r "PCC1_VZV_60_1_trimmed_P_1.fastq PCC1_VZV_60_1_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 1
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124839
   #Consensus Quality Before Polishing: 99.9992
   #Consensus QV Before Polishing: 50.96
   polca.sh -a PCC1_VZV_60_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa -r "PCC1_VZV_60_1_trimmed_P_1.fastq PCC1_VZV_60_1_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124839
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   
   #-- PCC1_VZV_60_4.assembly3-modify.fasta --
   polca.sh -a ../viralngs/tmp/02_assembly/PCC1_VZV_60_4.assembly3-modify.fasta -r "PCC1_VZV_60_4_trimmed_P_1.fastq PCC1_VZV_60_4_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 1
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124851
   #Consensus Quality Before Polishing: 99.9992
   #Consensus QV Before Polishing: 50.96
   polca.sh -a PCC1_VZV_60_4.assembly3-modify.fasta.PolcaCorrected.fa -r "PCC1_VZV_60_4_trimmed_P_1.fastq PCC1_VZV_60_4_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124851
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   
   #-- PCC1_VZV_60_6.assembly3-modify.fasta --
   polca.sh -a ../viralngs/tmp/02_assembly/PCC1_VZV_60_6.assembly3-modify.fasta -r "PCC1_VZV_60_6_trimmed_P_1.fastq PCC1_VZV_60_6_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 3
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124873
   #Consensus Quality Before Polishing: 99.9976
   #Consensus QV Before Polishing: 46.19
   polca.sh -a PCC1_VZV_60_6.assembly3-modify.fasta.PolcaCorrected.fa -r "PCC1_VZV_60_6_trimmed_P_1.fastq PCC1_VZV_60_6_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124871
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   

3. Multiple alignment of all corrected assembly

   cat ./20S_polished/VZV_20S.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa ./20c_polished/VZV_20c.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa ./20_1_polished/PCC1_VZV_20_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa ./20_2_polished/PCC1_VZV_20_2.assembly2-gapfilled.fasta.PolcaCorrected.fa.PolcaCorrected.fa ./20_5_polished/PCC1_VZV_20_5.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa > 20.fasta
   
   cat ./60S_polished/VZV_60S.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa ./60c_polished/VZV_60c.assembly2-gapfilled.fasta.PolcaCorrected.fa.PolcaCorrected.fa ./60_1_polished/PCC1_VZV_60_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa ./60_4_polished/PCC1_VZV_60_4.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa ./60_6_polished/PCC1_VZV_60_6.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa > 60.fasta
   
   mafft --clustalout 20.fasta > 20.aln
   mafft --clustalout 60.fasta > 60.aln
   
   grep "NC_001348.1_con" 20.aln > PCC1_VZV_20_2-1.fasta
   seqtk seq PCC1_VZV_20_2-1.fasta -l 60 > PCC1_VZV_20_2.fasta
   
   polca.sh -a PCC1_VZV_20_2.fasta -r "PCC1_VZV_20_2_trimmed_P_1.fastq PCC1_VZV_20_2_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124866
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   
   cat ./20S_polished/VZV_20S.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa ./20c_polished/VZV_20c.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa ./20_1_polished/PCC1_VZV_20_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa ./PCC1_VZV_20_2.fasta.PolcaCorrected.fa ./20_5_polished/PCC1_VZV_20_5.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa > 20_round2.fasta
   mafft --clustalout 20_round2.fasta > 20_round2.aln  #--leavegappyregion
   #(Optional) Delete "-1", set 2 positions of the lines "************"
   python check_SNP_positions.py
   
   grep "NC_001348.1_con" 60.aln > VZV_60c-1.fasta
   seqtk seq VZV_60c-1.fasta -l 60 > VZV_60c.fasta
   polca.sh -a VZV_60c.fasta -r "VZV_60c_trimmed_P_1.fastq VZV_60c_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 119660
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   cat ./60S_polished/VZV_60S.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa VZV_60c.fasta.PolcaCorrected.fa ./60_1_polished/PCC1_VZV_60_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa ./60_4_polished/PCC1_VZV_60_4.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa ./60_6_polished/PCC1_VZV_60_6.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa > 60_round2.fasta
   mafft --clustalout 60_round2.fasta > 60_round2.aln
   muscle -in 20_round2.fasta -out 20_round2.aln -clw
   #(Optional) Delete "-1", set 2 positions of the lines "************"
   python check_SNP_positions.py
   
   grep "VZV_60c-1" 60_round2.aln > VZV_60c-2.fasta
   seqtk seq VZV_60c-2.fasta -l 60 > VZV_60c-3.fasta
   polca.sh -a VZV_60c-3.fasta -r "VZV_60c_trimmed_P_1.fastq VZV_60c_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 119660
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   polca.sh -a VZV_60c-3.fasta.PolcaCorrected.fa -r "VZV_60c_trimmed_P_1.fastq VZV_60c_trimmed_P_2.fastq" -t 40 -m 10G
   #Stats BEFORE polishing:
   #Substitution Errors Found: 0
   #Insertion/Deletion Errors Found: 0
   #Assembly Size: 124896
   #Consensus Quality Before Polishing: 100
   #Consensus QV Before Polishing: 100.00
   cat ./60S_polished/VZV_60S.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa VZV_60c-3.fasta.PolcaCorrected.fa.PolcaCorrected.fa ./60_1_polished/PCC1_VZV_60_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa ./60_4_polished/PCC1_VZV_60_4.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa ./60_6_polished/PCC1_VZV_60_6.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa > 60_round3.fasta
   mafft --auto --op 3 --ep 0.1 --clustalout 60_round3.fasta > 60_round3.aln  #--leavegappyregion
   ulimit -s unlimited
   muscle -in 60_round3.fasta -out 60_round3.aln -clw -maxiters 2
   mamba install -c bioconda clustalo
   clustalo -i 60_round3.fasta -o output.fasta --auto
   mamba install -c bioconda t-coffee
   t_coffee -seq input.fasta -outfile output.fasta
   #(Optional) Delete "-1", set 2 positions of the lines "************"
   python check_SNP_positions.py
   
   1. Try a Different MAFFT Mode
       The default mode (--auto) may favor gaps too much. Use --globalpair or --localpair instead.
       Command:
       mafft --globalpair --maxiterate 1000 input.fasta > output.fasta
       or
       mafft --localpair --maxiterate 1000 input.fasta > output.fasta
           --globalpair: More accurate for closely related sequences.
           --localpair: Better if your sequences have recombination or partial homology.
   
   2. Increase Gap Open Penalty (--op)
       The default gap opening penalty is low, leading to excessive gaps.
       Try increasing it:
       mafft --auto --op 3 input.fasta > output.fasta
           Default is 1.53; higher values reduce gaps.
   
   3. Reduce Gap Extension Penalty (--ep)
       MAFFT extends gaps too easily. Lowering --ep discourages long gaps.
       mafft --auto --op 3 --ep 0.1 input.fasta > output.fasta
   
   cp ./20S_polished/VZV_20S.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
   cp ./20c_polished/VZV_20c.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
   cp ./20_1_polished/PCC1_VZV_20_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa toSend
   cp ./PCC1_VZV_20_2.fasta.PolcaCorrected.fa toSend
   cp ./20_5_polished/PCC1_VZV_20_5.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
   
   cp ./60S_polished/VZV_60S.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa toSend
   cp VZV_60c-3.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
   cp ./60_1_polished/PCC1_VZV_60_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa toSend
   cp ./60_4_polished/PCC1_VZV_60_4.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
   cp ./60_6_polished/PCC1_VZV_60_6.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
   

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