1. eDNA analyze
Wang Ying &
Huang Junman

2. Introduction Environmental DNA (eDNA)
trace DNA in samples such as water, soil, or faeces. eDNA is a mixture of potentially degraded DNA from many different organisms.

4. Sampling sites Shanghai Ocean University The East China Sea
Shanghai ocean aquarium
Yellow Mountain

5. Technology
1.Hybridization
2.PCR

6. MiFish，a set of universal PCR primers for metabarcoding environmental DNA from fishes :detection of more than 230 subtropical marine species

8. Data pre-processing MiSeq reads FASTQC, SUGAR Low-quality (out)
High-quality
SolexaQA
Tail-trimmed paired-end reads
FLASH
Assembled reads
Ambiguous sites
Unusual lengths
(out)
Perl
remove primer sequences
Expected size
( bp)
Pre-processed reads
TAGCLEANER
FASTQ
FASTA

9. Taxonomic assignment Pre-processed reads UCLUST “derep-fulllength”
>=10 identical reads
Under-represented sequences
Processed reads
Pairwise
If the identity more than or equal to 99%
UCLUST
“usearch-global”
BLASTN
Considered as idential
>=97%
E-value threshold of 10−5
Not the same species
<97%
The same species

10. Gene Bank https://www.ncbi.nlm.nih.gov/genome/browse/ Vertebrata
Organelles

11. Download NCBI gene bank
Smithwaterman
11 gene bank
Add flank sequence
Gene bank

12. Outline Database Original data Smithwaterman Trim getTarget
Rmrep(Remove replication)
Assembly
BLAST
Result
Selection

13. Read & contig

14. Trim & Rmrep
Trim low quality reads too short reads adapter Rmrep rmrep.pl

15. Assembly
Trinity
Abssy

16. getTarget seq
getarget.pl use 11 target seq of oreochromis to find target seq from database (120bp) + flank (300bp)

17. BLAST

18. E-value
The lower the E-value, or the closer it is to zero, the more "significant" the match is
the calculation of the E value takes into account the length of the query sequence
These high E values make sense because shorter sequences have a higher probability of occurring in the database purely by chance

19. Bit score
The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment. In general terms, this score is calculated from a formula that takes into account the alignment of similar or identical residues, as well as any gaps introduced to align the sequences.

20. Selection
Set threshold identity >97% Pick out maximum value of identity, e-value Pick out contig with more reads

21. Result identity e-value contig length number of reads Species name
Total number of species
Length of target seq & flank
Specie corresponding with which target seq (Tn)