MIDORI - MiFish-U

In-silico PCR

• Primer: MiFish-U
• Refernce: MIDORI based and fish related fragments

for E in {0..3}
do
  # PCR (exp size: 172nt without primer)
  ${u} -search_pcr MIDORI_FishHits.fa \
       -db Primer_MiFish.fa -strand both \
       -maxdiffs ${E} \
       -minamp 75 \
       -maxamp 275 \
       -pcrout MIDORI_MiFish_e${E}.hits \
       -ampout MIDORI_MiFish_e${E}.fa
done

PCR Fragment-Length Distribution:

# N(error0) =   248
# 210 ************************************************************ 178
# 220 ************************ 70

# N(error1) = 4,836
# 210 ************************************************************ 2,633
# 220 ************************************************* 2,163
# 230 * 38
# 240  0
# 250  1
# 260  1

# N(error2) = 5,759
# 210 ************************************************************ 3,162
# 220 *********************************************** 2,462
# 230 *** 133
# 240  0
# 250  1
# 260  1

# N(error3) = 6,349
# 210 ************************************************************ 3,347
# 220 ************************************************** 2,801
# 230 **** 199
# 240  0
# 250  1
# 260  1

# Note: error == mismatch

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"$12}' MIDORI_MiFish_e${E}.hits > MIDORI_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"$12}' MIDORI_MiFish_e${E}.hits > MIDORI_MiFish_e${E}_out.fa
done

Results (sequence counts):

## Clean Hits
cfa MIDORI_MiFish_e*_clean.fa
# MIDORI_MiFish_e0_clean.fa:   248
# MIDORI_MiFish_e1_clean.fa: 4,833
# MIDORI_MiFish_e2_clean.fa: 5,754
# MIDORI_MiFish_e3_clean.fa: 6,340

## Remove Species
cfa MIDORI_MiFish_e*_out.fa
# MIDORI_MiFish_e0_out.fa: 0
# MIDORI_MiFish_e1_out.fa: 3
# MIDORI_MiFish_e2_out.fa: 5
# MIDORI_MiFish_e3_out.fa: 9

## Removed Records (mismatches at the 3`-end):
* Species: Acrocheilus alutaceus (ID:67537)
* Species: Fonchiiloricaria nanodon (ID:912662)
* Species: Gobiomorus dormitor (ID:308076)
* Species: Gymnocypris potanini (ID:263516)
* Species: Harttiella lucifer (ID:1137733)
* Species: Kyphosus bigibbus (ID:990599)
* Species: Siniperca knerii (ID:214812)
* Species: Siniperca scherzeri (ID:228252)
* Species: Tetraodon lineatus (ID:1220758)

Summary

• MIDORI - MiFish-U

Step-by-step

for E in {0..3}
do
  # Class
  awk -F",c:" '{if($1 ~ />/) print $2}' MIDORI_MiFish_e${E}_clean.fa |\
  awk -F",|_" '{if(length($1)>0) print $1" (TaxID:"$2")"}' |\
  sort -u > MIDORI_MiFish_e${E}_Class.txt
  # Order
  awk -F",o:" '{if($1 ~ />/) print $2}' MIDORI_MiFish_e${E}_clean.fa |\
  awk -F",|_" '{if(length($1)>0) print $1" (TaxID:"$2")"}' |\
  sort -u > MIDORI_MiFish_e${E}_Order.txt
  # Family
  awk -F",f:" '{if($1 ~ />/) print $2}' MIDORI_MiFish_e${E}_clean.fa |\
  awk -F",|_" '{if(length($1)>0) print $1" (TaxID:"$2")"}' |\ 
  sort -u > MIDORI_MiFish_e${E}_Family.txt
  # Genus
  awk -F",g:" '{if($1 ~ />/) print $2}' MIDORI_MiFish_e${E}_clean.fa |\
  awk -F",|_" '{if(length($1)>0) print $1" (TaxID:"$2")"}' |\
  sort -u > MIDORI_MiFish_e${E}_Genus.txt
done
./PrintSummary.sh MIDORI_MiFish # Create Simple Summary Report including WebLogo