Cumaru-ferro

Laboratory Technical Information (BIO-TLI-ID-03-DO, v. 1.01, 2022-Jan-03):

Species identification

1.  trnH-psbA: intergenic spacer with enough variation at sequence level to identify samples of Dipteryx odorata at the species level
2. Expected fragment size: 450-630 bp
3. Foward primer: GTTATGCATGAACGTAATGCTC
4. Reverse primer: CGCGCATGGTGGATTCACAATCC
5. Reference: Mol Ecol (1998) 8: 513-525

Notes:

• BLAST search must be done at: http://idnaseq.biotool.com.br (authentication needed) using pre-defined parameters and selecting the specific database of the exploration permit (AUTEX or AUTEF).
• This procedure identify the sample at the species level and, sometimes, at the individual level (given the exploration permit’s DNA sequence database). However, if more than one tree is matched, individual identification is done by microsatellite sequencing.

Individual identification (if two or more trees are retrieved from the species identification procedure)

Notes:

• This is a step-by-step procedure, first sequence Do03, then Do08, and so on. Sometimes, multiple loci are needed to identify the individual tree.
• Microsatellite loci must be sequenced, do not use fragment length markers.

1. Microsatellite Do03:

1. Expected fragment size: 182-224 bp
2. Foward primer: AATTAGCCCAGTCCATAACA
3. Reverse primer: TGAGCTTCCAGAGCATGAGA
4. Reference: Mol Ecol Resour (1999) 9: 1542-1544

2. Microsatellite Do08:

1. Expected fragment size: 177-205 bp
2. Foward primer: AGATCAGCGGACAAAGGTCT
3. Reverse primer: GTAATGTTGTGCCACTCTTG
4. Reference: Mol Ecol Resour (1999) 9: 1542-1544

3. Microsatellite Do18:

1. Expected fragment size: 88-122 bp
2. Foward primer: GCTCTCTCCCCCTTTGTCTC
3. Reverse primer: GTTGGCAGTGAAGGTGGTG
4. Reference: Mol Ecol Resour (1999) 9: 1542-1544

4. Microsatellite Do19:

1. Expected fragment size: 109-141 bp
2. Foward primer: GCGTCTCTTCCCATTGTTTG
3. Reverse primer: TCCGTAGAGCCACACAGAGA
4. Reference: Mol Ecol Resour (1999) 9: 1542-1544

Sequence quality control (minimum): 

1. Sanger: phred value = 20
2. Illumina: depth = 10x
3. Oxford Nanopore: depth = 10x
4. Coverage: 90% of expected fragment size

Notes (Nat Rev Genet (2014) 15: 121–132):

• Sequencing depth can be defined theoretically as LN/G, where L is the read length, N is the number of reads and G is the haploid genome length.
• Sequencing coverage is the percentage of target bases that have been sequenced for a given number of times.