MitoFish - MiFish-U

In-silico PCR

• Primer: MiFish-u
• Reference: MitoFish based fragmments

for E in {0..3}
do
  ${u} -search_pcr MitoFish-Fragment_FRC.fa \
       -db Primer_MiFish.fa -strand both \
       -maxdiffs ${E} \
       -minamp 75 \
       -maxamp 275 \
       -pcrout MitoFish_MiFish_e${E}.hits \
       -ampout MitoFish_MiFish_e${E}.fa
done

PCR Fragment-Length Distribution:

# N(e0) = 281
# 210 ************************************************************ 198
# 220 ************************* 83
# 
# N(e1) = 7,039
# Display basic information about sequences
# 210 ************************************************************ 4,201
# 220 **************************************** 2,770
# 230 * 66
# 240  0
# 250  1
# 260  1
# 
# N(e2) = 8,203
# Display basic information about sequences
# 210 ************************************************************ 4,829
# 220 *************************************** 3,167
# 230 *** 205
# 240  0
# 250  1
# 260  1
# 
# N(e3) = 9,382
# Display basic information about sequences
# 210 ************************************************************ 5,066
# 220 ********************************************** 3,887
# 230 ***** 427
# 240  0
# 250  1
# 260  1

Problematic Hits

We remove PCR hits with primer mismatches at the last 2 positions at the 3`-end. This choice is based for reproducibility with Thang et al. (2020).

for E in {0..3}
do
  echo "Mis-Match: ${E}"
  awk -F"\t" '{if($5 == "MiFish-U-F" && $8 == "MiFish-U-R" && substr($7,length($7)-1,length($7)) !~ "[ATCG]" && substr($10,length($10)-1,length($10)) !~ "[ATCG]")  print ">"$1"\n"$12}' MitoFish_MiFish_e${E}.hits > MitoFish_MiFish_e${E}_clean.fa
  awk -F"\t" '{if($5 == "MiFish-U-F" && $8 == "MiFish-U-R" && (substr($7,length($7)-1,length($7)) ~ "[ATCG]" || substr($10,length($10)-1,length($10))  ~ "[ATCG]")) print ">"$1"\n"$12}' MitoFish_MiFish_e${E}.hits > MitoFish_MiFish_e${E}_out.fa
done

Results (sequence counts):

## Clean Hits
cfa MitoFish_MiFish_e*_clean.fa
# MitoFish_MiFish_e0_clean.fa:   281
# MitoFish_MiFish_e1_clean.fa: 7,036
# MitoFish_MiFish_e2_clean.fa: 8,197
# MitoFish_MiFish_e3_clean.fa: 9,371

## Remove Species
cfa MitoFish_MiFish_e*_out.fa
# MitoFish_MiFish_e0_out.fa:  0
# MitoFish_MiFish_e1_out.fa:  3
# MitoFish_MiFish_e2_out.fa:  6
# MitoFish_MiFish_e3_out.fa: 11

## Removed Records (mismatches at the 3`-end):
* Species: Acrocheilus alutaceus (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Fonchiiloricaria nanodon (["Siluriformes;"] ["Loricariidae;"]) 
* Species: Gobiomorus dormitor (["Gobiiformes;"] ["Eleotridae;"]) 
* Species: Gymnocypris potanini (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Harttiella lucifer (["Siluriformes;"] ["Loricariidae;"]) 
* Species: Kyphosus bigibbus (["Perciformes;"] ["Kyphosidae;"]) 
* Species: Piaractus sp. HC-2014 (["Characiformes;"] []) 
* Species: Siniperca knerii (["Centrarchiformes;"] ["Sinipercidae;"]) 
* Species: Siniperca scherzeri (["Centrarchiformes;"] ["Sinipercidae;"]) 
* Species: Tetraodon lineatus (["Tetraodontiformes;"] ["Tetraodontidae;"]) 

Summary

• MitoFish - MiFish