PIG (PIpeline for microsatellite Genotyping)

Experiment design

Samples are grouped to batches, which correspond to 96-well (number of wells can vary) sample plates. Each position on plate (=each sample) is marked by unique combination of tags (short oligoes, connected to primers). It allows us to sequence multiple samples during one run. The pipeline is developed to analize data from multiplex PCR, when all loci of interest are proceeded in a single reaction. After sequencing, we need to demultiplex all the samples and loci and then to perform allele calling. The main demultiplexing steps are based on OBITools3: https://git.metabarcoding.org/obitools/obitools3/blob/master/README.md

Input files

1. Sequences

Illumina paired-end reads in .fastq format.

2. Samples

Tab-sepatated text file.

Example:

Position	Sample	Type
A1	bear1	sample
A2	blank1	blank

Sample

Sample name. Please, avoid spaces, dots, backslashes and special symbols in sample names.

Postion

Enumerated by capital letters (rows) and numbers (columns). Typically, 96-well plate contains 8 rows and 12 columns. Each position is marked by a pair of tags (see Tags section).

Type:

• sample
• blank (empty well)
• PCRNeg (no template in PCR)
• ExtNeg (no sample in extraction)

3. Primers

Tab-sepatated text file. Type: microsat or snp. In case if the marker is biallelic or microsatellite contains different motifs, you need to provide all sequences or motifs with the same locus name. Sequence (in the "sequence" column) should be provided without primers.

Example:

locus	primerF	primerR	type	motif	sequence
UA_03	gctcccataac	gctcccataac	microsat	acac
ZF	cataacgctcc	taacgctcccataac	snp		agag........tatac

4. Tag combination file

Tab-sepatated text file. If you have the only one primer plate, call the column PP1 (primer plate 1).

Important. Make sure, that positions names in this file match to positions in the Sample description file.

Example:

Position	PP1	PP2
A1	acacacac:acacacac	caggctaa:tgagccta
B1	acacacac:acagcaca	caggctaa:tgagcctt
..	...	...
H12	gactgatg:gatcgcga	gaggacta:tcagtcga

5. Configuration file

Text file in .json format. "Library"

"Batches":

• Primers: path to the primer file
• Name: batch name
• Samples: path to the sample file
• Tag_plates: ["PP1", "PP2"] (list of tag combinations, used for the batch).
• Tags: path to the tag combination file

"Reads": path to the reads in fastq "Output_path": path to the output folder