Assembly correction tools: Polca, Pilon, and Medaka

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Tags: processing

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.

  1.     # Under the env (nextclade)
  2.     mamba install -c bioconda -c conda-forge masurca
  4.     #-- VZV_20S.assembly3-modify.fasta --
  5.     (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
  6.     #3
  7.     (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
  8.     #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.

  1.     bwa index PCC1_VZV_20_2.assembly3-modify.fasta.PolcaCorrected.fa
  2.     bwa mem -t 40 PCC1_VZV_20_2.assembly3-modify.fasta.PolcaCorrected.fa \
  3.         PCC1_VZV_20_2_trimmed_P_1.fastq \
  4.         PCC1_VZV_20_2_trimmed_P_2.fastq \
  5.         > aln.sam
  6.     samtools view -bS aln.sam > aln.bam
  7.     samtools sort aln.bam aln.sorted
  8.     samtools index aln.sorted.bam
  9.     (nextclade) pilon --genome PCC1_VZV_20_2.assembly3-modify.fasta.PolcaCorrected.fa \
  10.         --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

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

  2. Correcting assembly for Huang_Human_herpesvirus_3

    1. ./VZV_20S.fasta
    2. ./VZV_20c.fasta
    3. ./PCC1_VZV_20_1.fasta
    4. ./PCC1_VZV_20_2.fasta
    5. ./PCC1_VZV_20_5.fasta
    7. ./VZV_60S.fasta
    8. ./VZV_60c.fasta
    9. ./PCC1_VZV_60_1.fasta
    10. ./PCC1_VZV_60_4.fasta
    11. ./PCC1_VZV_60_6.fasta
    13. #find . -nma "*.assembly1-spades.fasta" | wc -l
    14. #find . -name "*.assembly2-gapfilled.fasta" | wc -l
    15. #find . -name "*.assembly3-modify.fasta" | wc -l
    16. #find . -name "*.assembly4-refined.fasta" | wc -l
    18. # Under the env (nextclade) and directory ~/DATA/Data_Huang_Human_herpesvirus_3/trimmed
    19. mamba install -c bioconda -c conda-forge masurca
    21. #-- VZV_20S.assembly3-modify.fasta --
    22. (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
    23. #Stats BEFORE polishing:
    24. #Substitution Errors Found: 3
    25. #Insertion/Deletion Errors Found: 0
    26. #Assembly Size: 124870
    27. #Consensus Quality Before Polishing: 99.9976
    28. #Consensus QV Before Polishing: 46.19
    29. (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
    30. #Stats BEFORE polishing:
    31. #Substitution Errors Found: 0
    32. #Insertion/Deletion Errors Found: 0
    33. #Assembly Size: 124869
    34. #Consensus Quality Before Polishing: 100
    35. #Consensus QV Before Polishing: 100.00
    37. #-- VZV_20c.assembly3-modify.fasta --
    38. 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
    39. #Stats BEFORE polishing:
    40. #Substitution Errors Found: 2
    41. #Insertion/Deletion Errors Found: 2
    42. #Assembly Size: 124872
    43. #Consensus Quality Before Polishing: 99.9968
    44. #Consensus QV Before Polishing: 44.94
    45. 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
    46. #Stats BEFORE polishing:
    47. #Substitution Errors Found: 0
    48. #Insertion/Deletion Errors Found: 0
    49. #Assembly Size: 124873
    50. #Consensus Quality Before Polishing: 100
    51. #Consensus QV Before Polishing: 100.00
    53. #-- PCC1_VZV_20_1.assembly3-modify.fasta --
    54. 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
    55. #Stats BEFORE polishing:
    56. #Substitution Errors Found: 1
    57. #Insertion/Deletion Errors Found: 1
    58. #Assembly Size: 124873
    59. #Consensus Quality Before Polishing: 99.9984
    60. #Consensus QV Before Polishing: 47.95
    61. 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
    62. #Stats BEFORE polishing:
    63. #Substitution Errors Found: 1
    64. #Insertion/Deletion Errors Found: 0
    65. #Assembly Size: 124873
    66. #Consensus Quality Before Polishing: 99.9992
    67. #Consensus QV Before Polishing: 50.96
    68. 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
    69. #Stats BEFORE polishing:
    70. #Substitution Errors Found: 0
    71. #Insertion/Deletion Errors Found: 0
    72. #Assembly Size: 124873
    73. #Consensus Quality Before Polishing: 100
    74. #Consensus QV Before Polishing: 100.00
    76. #-- PCC1_VZV_20_2.assembly3-modify.fasta --
    77. 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
    78. #Stats BEFORE polishing:
    79. #Substitution Errors Found: 1
    80. #Insertion/Deletion Errors Found: 1
    81. #Assembly Size: 124866
    82. #Consensus Quality Before Polishing: 99.9984
    83. #Consensus QV Before Polishing: 47.95
    84. 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
    85. #Stats BEFORE polishing:
    86. #Substitution Errors Found: 0
    87. #Insertion/Deletion Errors Found: 0
    88. #Assembly Size: 124867
    89. #Consensus Quality Before Polishing: 100
    90. #Consensus QV Before Polishing: 100.00
    92. #-- PCC1_VZV_20_5.assembly3-modify.fasta --
    93. 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
    94. #Stats BEFORE polishing:
    95. #Substitution Errors Found: 1
    96. #Insertion/Deletion Errors Found: 0
    97. #Assembly Size: 124873
    98. #Consensus Quality Before Polishing: 99.9992
    99. #Consensus QV Before Polishing: 50.96
    100. 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
    101. #Stats BEFORE polishing:
    102. #Substitution Errors Found: 0
    103. #Insertion/Deletion Errors Found: 0
    104. #Assembly Size: 124874
    105. #Consensus Quality Before Polishing: 100
    106. #Consensus QV Before Polishing: 100.00
    108. #-- VZV_60S.assembly3-modify.fasta --
    109. 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
    110. #Stats BEFORE polishing:
    111. #Substitution Errors Found: 2
    112. #Insertion/Deletion Errors Found: 0
    113. #Assembly Size: 124873
    114. #Consensus Quality Before Polishing: 99.9984
    115. #Consensus QV Before Polishing: 47.95
    116. 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
    117. #Stats BEFORE polishing:
    118. #Substitution Errors Found: 1
    119. #Insertion/Deletion Errors Found: 0
    120. #Assembly Size: 124870
    121. #Consensus Quality Before Polishing: 99.9992
    122. #Consensus QV Before Polishing: 50.96
    123. 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
    124. #Stats BEFORE polishing:
    125. #Substitution Errors Found: 0
    126. #Insertion/Deletion Errors Found: 0
    127. #Assembly Size: 124870
    128. #Consensus Quality Before Polishing: 100
    129. #Consensus QV Before Polishing: 100.00
    131. #-- VZV_60c.assembly2-gapfilled.fasta --
    132. 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
    133. #Stats BEFORE polishing:
    134. #Substitution Errors Found: 1
    135. #Insertion/Deletion Errors Found: 0
    136. #Assembly Size: 119660
    137. #Consensus Quality Before Polishing: 99.9992
    138. #Consensus QV Before Polishing: 50.78
    139. 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
    140. #Stats BEFORE polishing:
    141. #Substitution Errors Found: 0
    142. #Insertion/Deletion Errors Found: 0
    143. #Assembly Size: 119660
    144. #Consensus Quality Before Polishing: 100
    145. #Consensus QV Before Polishing: 100.00
    147. #-- PCC1_VZV_60_1.assembly3-modify.fasta --
    148. 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
    149. #Stats BEFORE polishing:
    150. #Substitution Errors Found: 0
    151. #Insertion/Deletion Errors Found: 0
    152. #Assembly Size: 124843
    153. #Consensus Quality Before Polishing: 100
    154. #Consensus QV Before Polishing: 100.00
    155. 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
    156. #Stats BEFORE polishing:
    157. #Substitution Errors Found: 1
    158. #Insertion/Deletion Errors Found: 0
    159. #Assembly Size: 124839
    160. #Consensus Quality Before Polishing: 99.9992
    161. #Consensus QV Before Polishing: 50.96
    162. 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
    163. #Stats BEFORE polishing:
    164. #Substitution Errors Found: 0
    165. #Insertion/Deletion Errors Found: 0
    166. #Assembly Size: 124839
    167. #Consensus Quality Before Polishing: 100
    168. #Consensus QV Before Polishing: 100.00
    170. #-- PCC1_VZV_60_4.assembly3-modify.fasta --
    171. 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
    172. #Stats BEFORE polishing:
    173. #Substitution Errors Found: 1
    174. #Insertion/Deletion Errors Found: 0
    175. #Assembly Size: 124851
    176. #Consensus Quality Before Polishing: 99.9992
    177. #Consensus QV Before Polishing: 50.96
    178. 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
    179. #Stats BEFORE polishing:
    180. #Substitution Errors Found: 0
    181. #Insertion/Deletion Errors Found: 0
    182. #Assembly Size: 124851
    183. #Consensus Quality Before Polishing: 100
    184. #Consensus QV Before Polishing: 100.00
    186. #-- PCC1_VZV_60_6.assembly3-modify.fasta --
    187. 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
    188. #Stats BEFORE polishing:
    189. #Substitution Errors Found: 3
    190. #Insertion/Deletion Errors Found: 0
    191. #Assembly Size: 124873
    192. #Consensus Quality Before Polishing: 99.9976
    193. #Consensus QV Before Polishing: 46.19
    194. 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
    195. #Stats BEFORE polishing:
    196. #Substitution Errors Found: 0
    197. #Insertion/Deletion Errors Found: 0
    198. #Assembly Size: 124871
    199. #Consensus Quality Before Polishing: 100
    200. #Consensus QV Before Polishing: 100.00

  3. Multiple alignment of all corrected assembly

    1. 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
    3. 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
    5. mafft --clustalout 20.fasta > 20.aln
    6. mafft --clustalout 60.fasta > 60.aln
    8. grep "NC_001348.1_con" 20.aln > PCC1_VZV_20_2-1.fasta
    9. seqtk seq PCC1_VZV_20_2-1.fasta -l 60 > PCC1_VZV_20_2.fasta
    11. 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
    12. #Stats BEFORE polishing:
    13. #Substitution Errors Found: 0
    14. #Insertion/Deletion Errors Found: 0
    15. #Assembly Size: 124866
    16. #Consensus Quality Before Polishing: 100
    17. #Consensus QV Before Polishing: 100.00
    19. 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
    20. mafft --clustalout 20_round2.fasta > 20_round2.aln  #--leavegappyregion
    21. #(Optional) Delete "-1", set 2 positions of the lines "************"
    22. python check_SNP_positions.py
    24. grep "NC_001348.1_con" 60.aln > VZV_60c-1.fasta
    25. seqtk seq VZV_60c-1.fasta -l 60 > VZV_60c.fasta
    26. polca.sh -a VZV_60c.fasta -r "VZV_60c_trimmed_P_1.fastq VZV_60c_trimmed_P_2.fastq" -t 40 -m 10G
    27. #Stats BEFORE polishing:
    28. #Substitution Errors Found: 0
    29. #Insertion/Deletion Errors Found: 0
    30. #Assembly Size: 119660
    31. #Consensus Quality Before Polishing: 100
    32. #Consensus QV Before Polishing: 100.00
    33. 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
    34. mafft --clustalout 60_round2.fasta > 60_round2.aln
    35. muscle -in 20_round2.fasta -out 20_round2.aln -clw
    36. #(Optional) Delete "-1", set 2 positions of the lines "************"
    37. python check_SNP_positions.py
    39. grep "VZV_60c-1" 60_round2.aln > VZV_60c-2.fasta
    40. seqtk seq VZV_60c-2.fasta -l 60 > VZV_60c-3.fasta
    41. polca.sh -a VZV_60c-3.fasta -r "VZV_60c_trimmed_P_1.fastq VZV_60c_trimmed_P_2.fastq" -t 40 -m 10G
    42. #Stats BEFORE polishing:
    43. #Substitution Errors Found: 0
    44. #Insertion/Deletion Errors Found: 0
    45. #Assembly Size: 119660
    46. #Consensus Quality Before Polishing: 100
    47. #Consensus QV Before Polishing: 100.00
    48. 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
    49. #Stats BEFORE polishing:
    50. #Substitution Errors Found: 0
    51. #Insertion/Deletion Errors Found: 0
    52. #Assembly Size: 124896
    53. #Consensus Quality Before Polishing: 100
    54. #Consensus QV Before Polishing: 100.00
    55. 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
    56. mafft --auto --op 3 --ep 0.1 --clustalout 60_round3.fasta > 60_round3.aln  #--leavegappyregion
    57. ulimit -s unlimited
    58. muscle -in 60_round3.fasta -out 60_round3.aln -clw -maxiters 2
    59. mamba install -c bioconda clustalo
    60. clustalo -i 60_round3.fasta -o output.fasta --auto
    61. mamba install -c bioconda t-coffee
    62. t_coffee -seq input.fasta -outfile output.fasta
    63. #(Optional) Delete "-1", set 2 positions of the lines "************"
    64. python check_SNP_positions.py
    66. 1. Try a Different MAFFT Mode
    67.     The default mode (--auto) may favor gaps too much. Use --globalpair or --localpair instead.
    68.     Command:
    69.     mafft --globalpair --maxiterate 1000 input.fasta > output.fasta
    70.     or
    71.     mafft --localpair --maxiterate 1000 input.fasta > output.fasta
    72.         --globalpair: More accurate for closely related sequences.
    73.         --localpair: Better if your sequences have recombination or partial homology.
    75. 2. Increase Gap Open Penalty (--op)
    76.     The default gap opening penalty is low, leading to excessive gaps.
    77.     Try increasing it:
    78.     mafft --auto --op 3 input.fasta > output.fasta
    79.         Default is 1.53; higher values reduce gaps.
    81. 3. Reduce Gap Extension Penalty (--ep)
    82.     MAFFT extends gaps too easily. Lowering --ep discourages long gaps.
    83.     mafft --auto --op 3 --ep 0.1 input.fasta > output.fasta
    85. cp ./20S_polished/VZV_20S.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
    86. cp ./20c_polished/VZV_20c.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
    87. cp ./20_1_polished/PCC1_VZV_20_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa toSend
    88. cp ./PCC1_VZV_20_2.fasta.PolcaCorrected.fa toSend
    89. cp ./20_5_polished/PCC1_VZV_20_5.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
    91. cp ./60S_polished/VZV_60S.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa toSend
    92. cp VZV_60c-3.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
    93. cp ./60_1_polished/PCC1_VZV_60_1.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa.PolcaCorrected.fa toSend
    94. cp ./60_4_polished/PCC1_VZV_60_4.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend
    95. cp ./60_6_polished/PCC1_VZV_60_6.assembly3-modify.fasta.PolcaCorrected.fa.PolcaCorrected.fa toSend

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