https://gatk.broadinstitute.org/hc/en-us/articles/360035531572-Evaluating-the-quality-of-a-germline-short-variant-callset

DNA Fingerprinting for Crop Varietal Identification:
Fit-for-Purpose Protocols, their Costs and Analytical
Implications

git clone https://github.91chi.fun//https://github.com/arq5x/bedtools2.git
cd bedtools2/
make

2.比对
使用bwa mem 比对每个样本（例如烟草的组装后的结果）

bwa mem ref.fa s1.fq | samtools sorted -o s1.sorted.bam -

3.过滤
过滤掉部分比对上的序列（soft clip和hard clip）和没有比对上的序列

samtools view s1.sorted.bam | awk '$6 !~ /H|S/{print}' | samtools view -bS - > s1.noclips.bam
samtools view -b -F 4  s1.noclips.bam -o s1.noclips.mapped.bam

4.取各个样本的交集
每次取2个 一直循环到最后1个

../bin/bedtools intersect -a s1.noclips.mapped.bam -b s2.noclips.mapped.bam -bed >intersect.s1.s2.bed
../bin/bedtools intersect -a intersect.s1.s2.bed -b s3.sorted.bam -bed >intersect.s1.s2.s3.bed

5.从每个样本提取序列
使用最终的交集 bed 文件 提取包含这些交集的reads 每个样本都取一次

samtools view s1.noclips.mapped.bam  -L intersect.s1.s2.s3.bed -o s1.regions.bam
samtools fasta s1.regions.bam -o s1.fasta

Sequences generated from the three PE libraries of B. nigra were assembled into contigs with MaSuRcA software (Zimin et al., 2013). Illumina PE reads were mapped on the raw Nanopore reads using BWA mem; 1 442 476 error corrected Nanopore reads were generated using the Pilon program (Walker et al., 2014). Scaffolding of the MaSuRcA based contigs with the error corrected Nanopore sequence data was carried out with SSPACE-LongRead.pl script (Boetzer et al., 2011), which generated 29 808 scaffolds with an N50 value of 19 834. Further, three rounds of scaffolding were carried out using the 2 kb, 6 kb and 10 kb MP library sequence data sets using SSPACE-STANDARD-3.0.pl script (Boetzer et al., 2011).

SNPs are more frequent than
SSRs in animal and plant genomes and the
mutation rate is lower, 10-8 (Kondrashov, 2003;
Nachman and Crowell, 2000) than in SSRs, 10-3
(Brinkmann et al., 1998); therefore, SNPs are
more reliable as molecular markers for organism
identification
SNP 比SSR有优势

cultivar determination of dried leaves is very
difficult because the original shape is changed
during the curing process and there are more
than 10,000 cultivars in existence.
用形态学鉴定干的叶子显然很困难 而且市面上有1万多个品种

从TGI获取的原始数据 用PCAP program做了组装
From the original 163,000 sequences, we assembled 31,370 contigs and about 60,000 singlets.

Primer Design and PCR Reaction
设计引物 然后扩增

Sequences were analyzed on
an A B I3 7 3 0 x l D N A A naly z er ( A p p lied
Biosystems, Foster City, CA).

The sequence
data from each amplicon were analyzed with
sequence analyzing software: CLC Workbench
(CLC Bio, Denmark), Clustal X (Larkin et al.,
2007), and SequencherTM 4 . 8 ( G ene Code
Corporation). The minimum and maximum PCR
p roduct length was 71 6 bp and 2, 047 bp ,
respectively, and the average length was 1,295
bp (Table 3). We removed the first 25 bp from
the sequence and compared only the first 26 bp
to 525 bp region.

找到的SNP

烟草的SNP频率不是很多

该文献对异源多倍体的snp分析对比了5种流程 可以重点看看