MitoFish - Teleo

In-silico PCR

• Primer: Teleo
• Reference: MitoFish based fragments

for E in {0..3}
do
  # PCR (exp size: 63nt)
  ${u} -search_pcr MitoFish-Fragment_FRC.fa \
       -db Primer_Teleo.fa -strand both \
       -maxdiffs ${E} \
       -minamp 20 \
       -maxamp 200 \
       -pcrout MitoFish_Teleo_e${E}.hits\
       -ampout MitoFish_Teleo_e${E}.fa
done

PCR Fragment-Length Distribution:

# N(e0) = 5,793
#  50  1
#  60  3
#  70  3
#  80  1
#  90 *********************************** 2,134
# 100 ************************************************************ 3,641
# 110  6
# 120  3
# 130  0
# 140  0
# 150  0
# 160  1

# N(e1) = 8,327
#  50  1
#  60  19
#  70  7
#  80  7
#  90 *********************************** 3,040
# 100 ************************************************************ 5,198
# 110  20
# 120  34
# 130  0
# 140  0
# 150  0
# 160  1

# N(e2) = 9,370
#  50  1
#  60  19
#  70  7
#  80  8
#  90 ****************************************** 3,829
# 100 ************************************************************ 5,451
# 110  20
# 120  34
# 130  0
# 140  0
# 150  0
# 160  1

# N(e3) = 9,423
#  50  1
#  60  19
#  70  7
#  80  8
#  90 ****************************************** 3,852
# 100 ************************************************************ 5,481
# 110  20
# 120  34
# 130  0
# 140  0
# 150  0
# 160  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 == "Teleo-F" && $8 == "Teleo-R" && substr($7,length($7)-1,length($7)) !~ "[ATCG]" && substr($10,length($10)-1,length($10)) !~ "[ATCG]")  print ">"$1"\n"$12}' MitoFish_Teleo_e${E}.hits > MitoFish_Teleo_e${E}_clean.fa
  awk -F"\t" '{if($5 == "Teleo-F" && $8 == "Teleo-R" && (substr($7,length($7)-1,length($7)) ~ "[ATCG]" || substr($10,length($10)-1,length($10))  ~ "[ATCG]")) print ">"$1"\n"$12}' MitoFish_Teleo_e${E}.hits > MitoFish_Teleo_e${E}_out.fa
done

Results (sequence counts):

## Clean Hits
cfa MitoFish_Teleo_e*_clean.fa
# MitoFish_Teleo_e0_clean.fa: 5,793
# MitoFish_Teleo_e1_clean.fa: 8,280
# MitoFish_Teleo_e2_clean.fa: 9,307
# MitoFish_Teleo_e3_clean.fa: 9,353

## Remove Species
cfa MitoFish_Teleo_e*_out.fa
# MitoFish_Teleo_e0_out.fa:  0
# MitoFish_Teleo_e1_out.fa: 47
# MitoFish_Teleo_e2_out.fa: 63
# MitoFish_Teleo_e3_out.fa: 70

## Removed Records (mismatches at the 3`-end):
* Species: Acanthaphritis unoorum (["Pempheriformes;"] ["Percophidae;"]) 
* Species: Amblydoras cf. affinis (["Siluriformes;"] ["Doradidae;"]) 
* Species: Amblygaster sirm (["Clupeiformes;"] ["Clupeidae;"]) 
* Species: Anguilla mossambica (["Anguilliformes;"] ["Anguillidae;"]) 
* Species: Apteronotus albifrons (["Gymnotiformes;"] ["Apteronotidae;"]) 
* Species: Apteronotus rostratus (["Gymnotiformes;"] ["Apteronotidae;"]) 
* Species: Ariosoma balearicum (["Anguilliformes;"] ["Congridae;"]) 
* Species: Barilius bendelisis (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Barilius malabaricus (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Betadevario ramachandrani (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Cabdio morar (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Callanthias japonicus ([] ["Callanthiidae;"]) 
* Species: Carassius carassius (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Chelon dumerili ([] ["Mugilidae;"]) 
* Species: Chelon planiceps ([] ["Mugilidae;"]) 
* Species: Cynoglossus puncticeps (["Pleuronectiformes;"] ["Cynoglossidae;"]) 
* Species: Danio dangila (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Danio margaritatus (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Danionella dracula (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Danio nigrofasciatus (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Danio rerio (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Danio rerio (["Cypriniformes;"] ["Danionidae;"]) 
* Species: Devario chrysotaeniatus (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Devario devario (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Devario laoensis (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Echiichthys vipera (["Perciformes;"] ["Trachinidae;"]) 
* Species: Electrona carlsbergi (["Myctophiformes;"] ["Myctophidae;"]) 
* Species: Esomus metallicus (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Etheostoma tuscumbia (["Perciformes;"] ["Percidae;"]) 
* Species: Ictalurus pricei (["Siluriformes;"] ["Ictaluridae;"]) 
* Species: Leptocypris sp. CBM ZF 11419 (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Luciosoma bleekeri (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Luciosoma setigerum (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Microdevario kubotai (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Microdevario nana (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Microrasbora erythromicron (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Microrasbora rubescens (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Nannocharax schoutedeni (["Characiformes;"] ["Distichodontidae;"]) 
* Species: Opsaridium microlepis (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Opsaridium ubangiense (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Oxydoras niger (["Siluriformes;"] ["Doradidae;"]) 
* Species: Paraplagusia bilineata (["Pleuronectiformes;"] ["Cynoglossidae;"]) 
* Species: Paraplagusia blochii (["Pleuronectiformes;"] ["Cynoglossidae;"]) 
* Species: Promethichthys prometheus (["Scombriformes;"] ["Gempylidae;"]) 
* Species: Raiamas buchholzi (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Raiamas guttatus (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Raiamas senegalensis (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Raiamas steindachneri (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Rineloricaria stewarti (["Siluriformes;"] ["Loricariidae;"]) 
* Species: Salmostoma bacalia (["Cypriniformes;"] ["Cyprinidae;"]) 
* Species: Sigmops gracilis (["Stomiiformes;"] ["Gonostomatidae;"]) 
* Species: Sillaginopsis panijus ([] ["Sillaginidae;"]) 
* Species: Squalomugil nasutus ([] ["Mugilidae;"]) 
* Species: Trachinus draco (["Perciformes;"] ["Trachinidae;"]) 

Summary

• MitoFish - Teleo