1. MiSeq 2x250 16S V4 下机测序数据经下面pipeline代码转换为OTU table
#!/bin/bash
if [ x$usearch == x ] ; then
echo Must set \$usearch >> /dev/stderr
exit 1
fi
rm -rf ../out
mkdir -p ../out
cd ../out
# Merge paired reads
# Add sample name to read label (-relabel option)
# Pool samples together in raw.fq (Linux cat command)
for Sample in Mock Soil Human Mouse
do
$usearch -fastq_mergepairs ../data/${Sample}*_R1.fq \
-fastqout $Sample.merged.fq -relabel $Sample.
cat $Sample.merged.fq >> all.merged.fq
done
# Strip primers (V4F is 19, V4R is 20)
$usearch -fastx_truncate all.merged.fq -stripleft 19 -stripright 20 \
-fastqout stripped.fq
# Quality filter
$usearch -fastq_filter stripped.fq -fastq_maxee 1.0 \
-fastaout filtered.fa -relabel Filt
# Find unique read sequences and abundances
$usearch -fastx_uniques filtered.fa -sizeout -relabel Uniq -fastaout uniques.fa
# Make 97% OTUs and filter chimeras
$usearch -cluster_otus uniques.fa -otus otus.fa -relabel Otu
# Denoise: predict biological sequences and filter chimeras
$usearch -unoise3 uniques.fa -zotus zotus.fa
##################################################
# Downstream analysis of OTU sequences & OTU table
# Can do this for both OTUs and ZOTUs, here do
# just OTUs to keep it simple.
##################################################
# Make OTU table
$usearch -otutab all.merged.fq -otus otus.fa -otutabout otutab_raw.txt
2. OTU table 经过 PICRUSt 工具 转换为 KEGG_ID table(表中的数字代表靶到该OTU或KEGG上的reads数量)
OTU_ID | Sample1 | Sample2 | Sample3 | Sample4 |
1 | 100 | 80 | 60 | 90 |
2 | 50 | 60 | 70 | 50 |
3 | 40 | 60 | 60 | 40 |
4 | 90 | 80 | 50 | 80 |
KEGG_ID | Sample1 | Sample2 | Sample3 | Sample4 |
ko1234 | 90 | 80 | 40 | 40 |
ko2556 | 60 | 60 | 50 | 10 |
ko6948 | 60 | 18 | 26 | 34 |
ko6549 | 66 | 93 | 10 | 80 |