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Renato Augusto Corrêa dos Santos edited this page May 22, 2024 · 55 revisions

Poster presentation - Institute of Plant Breeding, Genetics & Genomics (IPBGG) 2024 retreat

Here I (RACS) describe the analyses associated with the poster presentation on the IPBGG 2024 retreat entitled "Correlation analysis of paired leaf transcriptome and microbiome across diverse maize lines". Authors: Renato Augusto Correa dos Santos (corresponding author), Diego Mauricio Riano-Pachon and Jason G. Wallace.

Here is the poster file.

Here is the 2024-PBGG-Poster-Abstracts file

IPBGG retreat 2024

Computational resources

Part of the analyses were conducted on a local machine with Ubuntu 22 (Intel® Xeon(R) CPU E5-1620 v3 @ 3.50GHz × 8, with 64Gb RAM) and on Sapelo2 (GACRC, UGA), using the Slurm Workload Manager (therefore, some of the analyses below will have SLURM specific variables or commands, e.g. #SBATCH).

Description of datasets

16S dataset

Paired-end reads, 2x 200bp, were obtained on a Illumina HiSeq 2500.

Chloroplast-discriminating primers:

  • 799F (59-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACCMGGATTAGATACCCKG-39)
  • 1115R (59-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGGTTGCGCTCGTTG-39)

(they target the V5/V6 region of the gene)

They are mentioned in Wallace et al (2018) and comes from Shade et al. (2013).

Additional information can be found on ebi (biosample).

Results data associated with Wallace et al (2018) were downloaded from FigShare. Codes are also available in a Github repository.

RNAseq dataset

3′ mRNA sequencing method (QuantSeq, Lexogen GmBH) 90 nucleotide single-end reads using Illumina TruSeq primers on an Illumina NextSeq 500 with v2 chemistry.

Metadata for SRA associated with PRJNA383416 (Kremling et al. 2018) were first filtered to recover only samples from leaf - day and night (LMAD and LMAN).

Quality check

16S dataset

Running FastQC v0.11.9:

#!/bin/bash
#SBATCH --job-name=fastqc_wallace2018
#SBATCH --partition=batch
#SBATCH --ntasks=1
#SBATCH --cpus-per-task=1
#SBATCH --mem=1G
#SBATCH --time=1:00:00
#SBATCH --output=fastqc_%A-%a.out
#SBATCH --error=fastqc_%A-%a.err
#SBATCH [email protected]
#SBATCH --array=1-592
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

FASTQ1=`ls -1 /home/rc61809/16S_Wallace2018/RawReads/*_1.fastq.gz| head -n $SLURM_ARRAY_TASK_ID| tail -n1`
FASTQ2=`echo $FASTQ1 | sed 's/_1.fastq.gz/_2.fastq.gz/g'`
OUTDIR=`echo $FASTQ1 | cut -f6 -d'/' | sed 's/_1.fastq.gz//g' | sed 's/$/_fastqc/g'`

if [ ! -d $OUTDIR ]; then
  mkdir $OUTDIR
fi

mkdir $OUTDIR/{1,2}

ml FastQC/0.11.9-Java-11 # Load software module

fastqc --outdir "$OUTDIR"/1 $FASTQ1
fastqc --outdir "$OUTDIR"/2 $FASTQ2

MultiQC v1.21 was used to combine results of forward or reverse reads only in single reports.

RNAseq dataset

Running FastQC v0.11.9:

for FASTQ in $(ls *.fastq.gz);
 do
 echo $FASTQ;
 OUTDIR=`echo $FASTQ | sed 's/.fastq.gz/_fastqc/g'`;
 if [ ! -d $OUTDIR ];
  then
  mkdir $OUTDIR;
  fastqc -o $OUTDIR $FASTQ;
 fi;
done;

Quality trimming

16S dataset - Cutadapt

Given that we observed low quality in general for in FastQC results, we decided to proceed to downstream analyses with the Forward reads only. Cutadapt v4.5 was used to trim using minimum quality threshold of 20 in both read sides and a minimum length of 150bp for forward reads.

#!/bin/bash
#SBATCH --job-name=cutadapt_wallace2018_q20_150bp
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=8
#SBATCH --ntasks-per-node=8
#SBATCH --cpus-per-task=1
#SBATCH --mem=64G
#SBATCH --time=10:00:00
#SBATCH --output=cutadapt_q20_150bp_%A-%a.out
#SBATCH --error=cutadapt_q20_150bp_%A-%a.err
#SBATCH [email protected]
#SBATCH --array=1-592
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

FASTQ1=`ls -1 /home/rc61809/16S_Wallace2018/RawReads/*_1.fastq.gz| head -n $SLURM_ARRAY_TASK_ID| tail -n1`
FASTQ2=`echo $FASTQ1 | sed 's/_1.fastq.gz/_2.fastq.gz/g'`
OUTDIR=`echo $FASTQ1 | cut -f6 -d'/' | sed 's/_1.fastq.gz//g' | sed 's/$/_cutadapt/g'`
OUTFASTQ1=`echo $FASTQ1 | cut -f6 -d'/'| sed 's/_1.fastq.gz/.out_1.fastq.gz/g'`
OUTFASTQ2=`echo $FASTQ2 | cut -f6 -d'/'| sed 's/_2.fastq.gz/.out_2.fastq.gz/g'`

if [ ! -d $OUTDIR ]; then
  mkdir $OUTDIR
fi

ml cutadapt/4.5-GCCcore-11.3.0  # Load software module

cutadapt -q 20,20 -m 150:0 --cores=8 -g TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -G GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -a CTGTCTCTTATA -A CTGTCTCTTATA -o $OUTDIR/$OUTFASTQ1 -p $OUTDIR/$OUTFASTQ2 $FASTQ1 $FASTQ2

RNAseq dataset - TrimGalore

RNAseq was cleaned with TrimGalore v0.6.10:

for FASTQ in $(ls *.fastq.gz);
 do echo $FASTQ;
 PREFIX=`echo $FASTQ | sed 's/.fastq.gz//g'` ;
 OUTDIR=`echo $FASTQ | sed 's/.fastq.gz/_trim_galore/g'`;
 if [ ! -d $OUTDIR ]; then
  trim_galore --output_dir $OUTDIR --basename $PREFIX --cores 1 $FASTQ;
 fi;
done;

Quantifying transcript expression

Expression was quantified against the transcriptome derived from representative transcripts of Maize v5 from Phytozome. The original file (Zmays_833_Zm-B73-REFERENCE-NAM-5.0.55.transcript_primaryTranscriptOnly.fa.gz) was modified to leave only the transcript identifiers on FASTA headers (without descriptions).

Indexing the transcriptome

salmon index --transcripts Zma2_rnas.fa --index index # maize v5

Quantifying transcript expression

Using the trimmed reads from TrimGalore to quantify expression in salmon v1.9:

for SAMPLE in $(ls *trim_galore/*_trimmed.fq.gz);
 do
 OUTDIR=`echo $SAMPLE | cut -f1 -d'/' | sed 's/_trim_galore/_Zma2_quant/g'`;
 salmon quant --threads 6 -i /media/renato/SSD1TB/Projects/UGA_BEPE_RenatoSantos/RNAseq/maize_transcriptomes/Zmays_Zm_B73_REFERENCE_NAM_5_0_55/index -l A -r "$SAMPLE" -o "$OUTDIR"
done;

Generating the counts and TPM matrices with quantmerge:

#!/bin/bash
#SBATCH --job-name=salmon_quantmerge_kremling2018
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --ntasks-per-node=1
#SBATCH --cpus-per-task=1
#SBATCH --mem=1G
#SBATCH --time=1:00:00
#SBATCH --output=salmon_quantmerge_Zma2.out
#SBATCH --error=salmon_quantmerge_Zma2.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml Salmon/1.9.0-GCC-11.3.0 # Load software module

cd /home/rc61809/Kremling_RNAseq

salmon quantmerge --quants SRR5909626_Zma2_quant SRR5909627_Zma2_quant SRR5909633_Zma2_quant SRR5909635_Zma2_quant SRR5909639_Zma2_quant SRR5909642_Zma2_quant SRR5909645_Zma2_quant SRR5909653_Zma2_quant SRR5909655_Zma2_quant SRR5909665_Zma2_quant SRR5909671_Zma2_quant SRR5909672_Zma2_quant SRR5909678_Zma2_quant SRR5909679_Zma2_quant SRR5909687_Zma2_quant SRR5909690_Zma2_quant SRR5909698_Zma2_quant SRR5909699_Zma2_quant SRR5909713_Zma2_quant SRR5909716_Zma2_quant SRR5909721_Zma2_quant SRR5909726_Zma2_quant SRR5909732_Zma2_quant SRR5909739_Zma2_quant SRR5909744_Zma2_quant SRR5909745_Zma2_quant SRR5909752_Zma2_quant SRR5909753_Zma2_quant SRR5909766_Zma2_quant SRR5909774_Zma2_quant SRR5909775_Zma2_quant SRR5909778_Zma2_quant SRR5909782_Zma2_quant SRR5909788_Zma2_quant SRR5909789_Zma2_quant SRR5909792_Zma2_quant SRR5909795_Zma2_quant SRR5909800_Zma2_quant SRR5909806_Zma2_quant SRR5909807_Zma2_quant SRR5909813_Zma2_quant SRR5909814_Zma2_quant SRR5909818_Zma2_quant SRR5909820_Zma2_quant SRR5909823_Zma2_quant SRR5909826_Zma2_quant SRR5909841_Zma2_quant SRR5909861_Zma2_quant SRR5909864_Zma2_quant SRR5909865_Zma2_quant SRR5909866_Zma2_quant SRR5909875_Zma2_quant SRR5909877_Zma2_quant SRR5909882_Zma2_quant SRR5909883_Zma2_quant SRR5909888_Zma2_quant SRR5909891_Zma2_quant SRR5909901_Zma2_quant SRR5909904_Zma2_quant SRR5909907_Zma2_quant SRR5909909_Zma2_quant SRR5909910_Zma2_quant SRR5909911_Zma2_quant SRR5909913_Zma2_quant SRR5909917_Zma2_quant SRR5909918_Zma2_quant SRR5909919_Zma2_quant SRR5909922_Zma2_quant SRR5909930_Zma2_quant SRR5909931_Zma2_quant SRR5909936_Zma2_quant SRR5909939_Zma2_quant SRR5909942_Zma2_quant SRR5909943_Zma2_quant SRR5909948_Zma2_quant SRR5909951_Zma2_quant SRR5909952_Zma2_quant SRR5909959_Zma2_quant SRR5909964_Zma2_quant SRR5909965_Zma2_quant SRR5909972_Zma2_quant SRR5909973_Zma2_quant SRR5909974_Zma2_quant SRR5909997_Zma2_quant SRR5910013_Zma2_quant SRR5910014_Zma2_quant SRR5910015_Zma2_quant SRR5910020_Zma2_quant SRR5910021_Zma2_quant SRR5910037_Zma2_quant SRR5910039_Zma2_quant SRR5910043_Zma2_quant SRR5910044_Zma2_quant SRR5910051_Zma2_quant SRR5910052_Zma2_quant SRR5910054_Zma2_quant SRR5910059_Zma2_quant SRR5910061_Zma2_quant SRR5910062_Zma2_quant SRR5910064_Zma2_quant SRR5910065_Zma2_quant SRR5910066_Zma2_quant SRR5910067_Zma2_quant SRR5910068_Zma2_quant SRR5910069_Zma2_quant SRR5910070_Zma2_quant SRR5910071_Zma2_quant SRR5910072_Zma2_quant SRR5910076_Zma2_quant SRR5910084_Zma2_quant SRR5910087_Zma2_quant SRR5910094_Zma2_quant SRR5910102_Zma2_quant SRR5910103_Zma2_quant SRR5910104_Zma2_quant SRR5910116_Zma2_quant SRR5910117_Zma2_quant SRR5910121_Zma2_quant SRR5910122_Zma2_quant SRR5910658_Zma2_quant SRR5910659_Zma2_quant SRR5910663_Zma2_quant SRR5910664_Zma2_quant SRR5910665_Zma2_quant SRR5910668_Zma2_quant SRR5910676_Zma2_quant SRR5910677_Zma2_quant SRR5910679_Zma2_quant SRR5910688_Zma2_quant SRR5910689_Zma2_quant SRR5910694_Zma2_quant SRR5910695_Zma2_quant SRR5910697_Zma2_quant SRR5910704_Zma2_quant SRR5910705_Zma2_quant SRR5910715_Zma2_quant SRR5910718_Zma2_quant SRR5910721_Zma2_quant SRR5910723_Zma2_quant SRR5910724_Zma2_quant SRR5910730_Zma2_quant SRR5910731_Zma2_quant SRR5910732_Zma2_quant SRR5910735_Zma2_quant SRR5910737_Zma2_quant SRR5910738_Zma2_quant SRR5910739_Zma2_quant SRR5910740_Zma2_quant SRR5910749_Zma2_quant SRR5910757_Zma2_quant SRR5910759_Zma2_quant SRR5910760_Zma2_quant SRR5910766_Zma2_quant SRR5910767_Zma2_quant SRR5910771_Zma2_quant SRR5910772_Zma2_quant SRR5910774_Zma2_quant SRR5910779_Zma2_quant SRR5910783_Zma2_quant SRR5910786_Zma2_quant SRR5910788_Zma2_quant SRR5910791_Zma2_quant SRR5910802_Zma2_quant SRR5910805_Zma2_quant SRR5910806_Zma2_quant SRR5910815_Zma2_quant SRR5910817_Zma2_quant SRR5910822_Zma2_quant SRR5910823_Zma2_quant SRR5910829_Zma2_quant SRR5910830_Zma2_quant SRR5910835_Zma2_quant SRR5910843_Zma2_quant SRR5910851_Zma2_quant SRR5910854_Zma2_quant SRR5910855_Zma2_quant SRR5910862_Zma2_quant SRR5910867_Zma2_quant SRR5910868_Zma2_quant SRR5910877_Zma2_quant SRR5910878_Zma2_quant SRR5910881_Zma2_quant SRR5910882_Zma2_quant SRR5910883_Zma2_quant SRR5910890_Zma2_quant SRR5910895_Zma2_quant SRR5910902_Zma2_quant SRR5910908_Zma2_quant SRR5910909_Zma2_quant SRR5910915_Zma2_quant SRR5910916_Zma2_quant SRR5910925_Zma2_quant SRR5910930_Zma2_quant SRR5910935_Zma2_quant SRR5910936_Zma2_quant SRR5910941_Zma2_quant SRR5910943_Zma2_quant SRR5910949_Zma2_quant SRR5910955_Zma2_quant SRR5910956_Zma2_quant SRR5910967_Zma2_quant SRR5910970_Zma2_quant SRR5910971_Zma2_quant SRR5910972_Zma2_quant SRR5910973_Zma2_quant SRR5910981_Zma2_quant SRR5910982_Zma2_quant SRR5910986_Zma2_quant SRR5910993_Zma2_quant SRR5910996_Zma2_quant SRR5910997_Zma2_quant SRR5911002_Zma2_quant SRR5911004_Zma2_quant SRR5911008_Zma2_quant SRR5911011_Zma2_quant SRR5911014_Zma2_quant SRR5911020_Zma2_quant SRR5911023_Zma2_quant SRR5911032_Zma2_quant SRR5911035_Zma2_quant SRR5911043_Zma2_quant SRR5911044_Zma2_quant SRR5911045_Zma2_quant SRR5911046_Zma2_quant SRR5911051_Zma2_quant SRR5911054_Zma2_quant SRR5911057_Zma2_quant SRR5911063_Zma2_quant SRR5911064_Zma2_quant SRR5911065_Zma2_quant SRR5911068_Zma2_quant SRR5911070_Zma2_quant SRR5911080_Zma2_quant SRR5911081_Zma2_quant SRR5911088_Zma2_quant SRR5911089_Zma2_quant SRR5911093_Zma2_quant SRR5911099_Zma2_quant SRR5911108_Zma2_quant SRR5911111_Zma2_quant SRR5911117_Zma2_quant SRR5911122_Zma2_quant SRR5911125_Zma2_quant SRR5911128_Zma2_quant SRR5911129_Zma2_quant SRR5911136_Zma2_quant SRR5911139_Zma2_quant SRR5911145_Zma2_quant SRR5911146_Zma2_quant SRR5911148_Zma2_quant SRR5911160_Zma2_quant SRR5911163_Zma2_quant SRR5911165_Zma2_quant SRR5911172_Zma2_quant SRR5911173_Zma2_quant SRR5911182_Zma2_quant SRR5911184_Zma2_quant SRR5911185_Zma2_quant SRR5911198_Zma2_quant SRR5911203_Zma2_quant SRR5911212_Zma2_quant SRR5911214_Zma2_quant SRR5911219_Zma2_quant SRR5911220_Zma2_quant SRR5911222_Zma2_quant SRR5911227_Zma2_quant SRR5911229_Zma2_quant SRR5911231_Zma2_quant SRR5911236_Zma2_quant SRR5911237_Zma2_quant SRR5911247_Zma2_quant SRR5911248_Zma2_quant SRR5911251_Zma2_quant SRR5911258_Zma2_quant SRR5911259_Zma2_quant SRR5911265_Zma2_quant SRR5911266_Zma2_quant SRR5911277_Zma2_quant SRR5911278_Zma2_quant SRR5911284_Zma2_quant SRR5911290_Zma2_quant SRR5911291_Zma2_quant SRR5911298_Zma2_quant SRR5911302_Zma2_quant SRR5911309_Zma2_quant SRR5911310_Zma2_quant SRR5911312_Zma2_quant SRR5911326_Zma2_quant SRR5911327_Zma2_quant SRR5911329_Zma2_quant SRR5911330_Zma2_quant SRR5911333_Zma2_quant SRR5911337_Zma2_quant SRR5911344_Zma2_quant SRR5911345_Zma2_quant SRR5911347_Zma2_quant SRR5911350_Zma2_quant SRR5911357_Zma2_quant SRR5911360_Zma2_quant SRR5911363_Zma2_quant SRR5911370_Zma2_quant SRR5911375_Zma2_quant SRR5911376_Zma2_quant SRR5911392_Zma2_quant SRR5911393_Zma2_quant SRR5911396_Zma2_quant SRR5911398_Zma2_quant SRR5911399_Zma2_quant SRR5911421_Zma2_quant SRR5911426_Zma2_quant SRR5911427_Zma2_quant SRR5911433_Zma2_quant SRR5911436_Zma2_quant SRR5911437_Zma2_quant SRR5911443_Zma2_quant SRR5911444_Zma2_quant SRR5911448_Zma2_quant SRR5911452_Zma2_quant SRR5911458_Zma2_quant SRR5911459_Zma2_quant SRR5911464_Zma2_quant SRR5911469_Zma2_quant SRR5911472_Zma2_quant SRR5911475_Zma2_quant SRR5911477_Zma2_quant SRR5911479_Zma2_quant SRR5911490_Zma2_quant SRR5911491_Zma2_quant SRR5911494_Zma2_quant SRR5911497_Zma2_quant SRR5911502_Zma2_quant SRR5911504_Zma2_quant SRR5911512_Zma2_quant SRR5911518_Zma2_quant SRR5911519_Zma2_quant SRR5911524_Zma2_quant SRR5911532_Zma2_quant SRR5911534_Zma2_quant SRR5911535_Zma2_quant SRR5911537_Zma2_quant SRR5911538_Zma2_quant SRR5911542_Zma2_quant SRR5911544_Zma2_quant SRR5911547_Zma2_quant SRR5911548_Zma2_quant SRR5911549_Zma2_quant SRR5911552_Zma2_quant SRR5911554_Zma2_quant SRR5911560_Zma2_quant SRR5911566_Zma2_quant SRR5911571_Zma2_quant SRR5911573_Zma2_quant SRR5911578_Zma2_quant SRR5911581_Zma2_quant SRR5911582_Zma2_quant SRR5911587_Zma2_quant SRR5911590_Zma2_quant SRR5911593_Zma2_quant SRR5911595_Zma2_quant SRR5911597_Zma2_quant SRR5911602_Zma2_quant SRR5911605_Zma2_quant SRR5911606_Zma2_quant SRR5911607_Zma2_quant SRR5911608_Zma2_quant SRR5911621_Zma2_quant SRR5911623_Zma2_quant SRR5911628_Zma2_quant SRR5911636_Zma2_quant SRR5911644_Zma2_quant SRR5911646_Zma2_quant SRR5911649_Zma2_quant SRR5911653_Zma2_quant SRR5911654_Zma2_quant SRR5911660_Zma2_quant SRR5911663_Zma2_quant SRR5911664_Zma2_quant SRR5911670_Zma2_quant SRR5911680_Zma2_quant SRR5911681_Zma2_quant SRR5911695_Zma2_quant SRR5911696_Zma2_quant SRR5911698_Zma2_quant SRR5911705_Zma2_quant SRR5911706_Zma2_quant SRR5911714_Zma2_quant SRR5911718_Zma2_quant SRR5911721_Zma2_quant SRR5911726_Zma2_quant SRR5911729_Zma2_quant SRR5911731_Zma2_quant SRR5911739_Zma2_quant SRR5911749_Zma2_quant SRR5911751_Zma2_quant SRR5911765_Zma2_quant SRR5911770_Zma2_quant SRR5911774_Zma2_quant SRR5911783_Zma2_quant SRR5911784_Zma2_quant SRR5911786_Zma2_quant SRR5911787_Zma2_quant SRR5911788_Zma2_quant SRR5911789_Zma2_quant SRR5911794_Zma2_quant SRR5911797_Zma2_quant SRR5911807_Zma2_quant SRR5911812_Zma2_quant SRR5911819_Zma2_quant SRR5911820_Zma2_quant SRR5911822_Zma2_quant SRR5911824_Zma2_quant SRR5911827_Zma2_quant SRR5911832_Zma2_quant SRR5911833_Zma2_quant SRR5911838_Zma2_quant SRR5911841_Zma2_quant SRR5911844_Zma2_quant SRR5911845_Zma2_quant SRR5911846_Zma2_quant SRR5911849_Zma2_quant SRR5911857_Zma2_quant SRR5911860_Zma2_quant SRR5911861_Zma2_quant SRR5911864_Zma2_quant SRR5911872_Zma2_quant SRR5911876_Zma2_quant SRR5911892_Zma2_quant SRR5911898_Zma2_quant SRR5911899_Zma2_quant SRR5911905_Zma2_quant SRR5911906_Zma2_quant SRR5911908_Zma2_quant SRR5911909_Zma2_quant SRR5911915_Zma2_quant SRR5911916_Zma2_quant SRR5911922_Zma2_quant SRR5911928_Zma2_quant SRR5911929_Zma2_quant SRR5911930_Zma2_quant SRR5911940_Zma2_quant SRR5911941_Zma2_quant SRR5911943_Zma2_quant SRR5911944_Zma2_quant SRR5911946_Zma2_quant SRR5911947_Zma2_quant SRR5911953_Zma2_quant SRR5911955_Zma2_quant SRR5911956_Zma2_quant SRR5911968_Zma2_quant SRR5911971_Zma2_quant SRR5911982_Zma2_quant SRR5911987_Zma2_quant SRR5911988_Zma2_quant SRR5911991_Zma2_quant SRR5911992_Zma2_quant SRR5911993_Zma2_quant SRR5911998_Zma2_quant SRR5912005_Zma2_quant SRR5912006_Zma2_quant SRR5912009_Zma2_quant SRR5912014_Zma2_quant SRR5912015_Zma2_quant SRR5912028_Zma2_quant SRR5912029_Zma2_quant SRR5912032_Zma2_quant SRR5912036_Zma2_quant SRR5912037_Zma2_quant SRR5912039_Zma2_quant SRR5912043_Zma2_quant SRR5912044_Zma2_quant SRR5912053_Zma2_quant SRR5912054_Zma2_quant SRR5912061_Zma2_quant SRR5912065_Zma2_quant SRR5912066_Zma2_quant SRR5912072_Zma2_quant SRR5912073_Zma2_quant SRR5912081_Zma2_quant SRR5912082_Zma2_quant SRR5912083_Zma2_quant SRR5912093_Zma2_quant SRR5912094_Zma2_quant SRR5912104_Zma2_quant SRR5912105_Zma2_quant SRR5912111_Zma2_quant SRR5912116_Zma2_quant --names SRR5909626 SRR5909627 SRR5909633 SRR5909635 SRR5909639 SRR5909642 SRR5909645 SRR5909653 SRR5909655 SRR5909665 SRR5909671 SRR5909672 SRR5909678 SRR5909679 SRR5909687 SRR5909690 SRR5909698 SRR5909699 SRR5909713 SRR5909716 SRR5909721 SRR5909726 SRR5909732 SRR5909739 SRR5909744 SRR5909745 SRR5909752 SRR5909753 SRR5909766 SRR5909774 SRR5909775 SRR5909778 SRR5909782 SRR5909788 SRR5909789 SRR5909792 SRR5909795 SRR5909800 SRR5909806 SRR5909807 SRR5909813 SRR5909814 SRR5909818 SRR5909820 SRR5909823 SRR5909826 SRR5909841 SRR5909861 SRR5909864 SRR5909865 SRR5909866 SRR5909875 SRR5909877 SRR5909882 SRR5909883 SRR5909888 SRR5909891 SRR5909901 SRR5909904 SRR5909907 SRR5909909 SRR5909910 SRR5909911 SRR5909913 SRR5909917 SRR5909918 SRR5909919 SRR5909922 SRR5909930 SRR5909931 SRR5909936 SRR5909939 SRR5909942 SRR5909943 SRR5909948 SRR5909951 SRR5909952 SRR5909959 SRR5909964 SRR5909965 SRR5909972 SRR5909973 SRR5909974 SRR5909997 SRR5910013 SRR5910014 SRR5910015 SRR5910020 SRR5910021 SRR5910037 SRR5910039 SRR5910043 SRR5910044 SRR5910051 SRR5910052 SRR5910054 SRR5910059 SRR5910061 SRR5910062 SRR5910064 SRR5910065 SRR5910066 SRR5910067 SRR5910068 SRR5910069 SRR5910070 SRR5910071 SRR5910072 SRR5910076 SRR5910084 SRR5910087 SRR5910094 SRR5910102 SRR5910103 SRR5910104 SRR5910116 SRR5910117 SRR5910121 SRR5910122 SRR5910658 SRR5910659 SRR5910663 SRR5910664 SRR5910665 SRR5910668 SRR5910676 SRR5910677 SRR5910679 SRR5910688 SRR5910689 SRR5910694 SRR5910695 SRR5910697 SRR5910704 SRR5910705 SRR5910715 SRR5910718 SRR5910721 SRR5910723 SRR5910724 SRR5910730 SRR5910731 SRR5910732 SRR5910735 SRR5910737 SRR5910738 SRR5910739 SRR5910740 SRR5910749 SRR5910757 SRR5910759 SRR5910760 SRR5910766 SRR5910767 SRR5910771 SRR5910772 SRR5910774 SRR5910779 SRR5910783 SRR5910786 SRR5910788 SRR5910791 SRR5910802 SRR5910805 SRR5910806 SRR5910815 SRR5910817 SRR5910822 SRR5910823 SRR5910829 SRR5910830 SRR5910835 SRR5910843 SRR5910851 SRR5910854 SRR5910855 SRR5910862 SRR5910867 SRR5910868 SRR5910877 SRR5910878 SRR5910881 SRR5910882 SRR5910883 SRR5910890 SRR5910895 SRR5910902 SRR5910908 SRR5910909 SRR5910915 SRR5910916 SRR5910925 SRR5910930 SRR5910935 SRR5910936 SRR5910941 SRR5910943 SRR5910949 SRR5910955 SRR5910956 SRR5910967 SRR5910970 SRR5910971 SRR5910972 SRR5910973 SRR5910981 SRR5910982 SRR5910986 SRR5910993 SRR5910996 SRR5910997 SRR5911002 SRR5911004 SRR5911008 SRR5911011 SRR5911014 SRR5911020 SRR5911023 SRR5911032 SRR5911035 SRR5911043 SRR5911044 SRR5911045 SRR5911046 SRR5911051 SRR5911054 SRR5911057 SRR5911063 SRR5911064 SRR5911065 SRR5911068 SRR5911070 SRR5911080 SRR5911081 SRR5911088 SRR5911089 SRR5911093 SRR5911099 SRR5911108 SRR5911111 SRR5911117 SRR5911122 SRR5911125 SRR5911128 SRR5911129 SRR5911136 SRR5911139 SRR5911145 SRR5911146 SRR5911148 SRR5911160 SRR5911163 SRR5911165 SRR5911172 SRR5911173 SRR5911182 SRR5911184 SRR5911185 SRR5911198 SRR5911203 SRR5911212 SRR5911214 SRR5911219 SRR5911220 SRR5911222 SRR5911227 SRR5911229 SRR5911231 SRR5911236 SRR5911237 SRR5911247 SRR5911248 SRR5911251 SRR5911258 SRR5911259 SRR5911265 SRR5911266 SRR5911277 SRR5911278 SRR5911284 SRR5911290 SRR5911291 SRR5911298 SRR5911302 SRR5911309 SRR5911310 SRR5911312 SRR5911326 SRR5911327 SRR5911329 SRR5911330 SRR5911333 SRR5911337 SRR5911344 SRR5911345 SRR5911347 SRR5911350 SRR5911357 SRR5911360 SRR5911363 SRR5911370 SRR5911375 SRR5911376 SRR5911392 SRR5911393 SRR5911396 SRR5911398 SRR5911399 SRR5911421 SRR5911426 SRR5911427 SRR5911433 SRR5911436 SRR5911437 SRR5911443 SRR5911444 SRR5911448 SRR5911452 SRR5911458 SRR5911459 SRR5911464 SRR5911469 SRR5911472 SRR5911475 SRR5911477 SRR5911479 SRR5911490 SRR5911491 SRR5911494 SRR5911497 SRR5911502 SRR5911504 SRR5911512 SRR5911518 SRR5911519 SRR5911524 SRR5911532 SRR5911534 SRR5911535 SRR5911537 SRR5911538 SRR5911542 SRR5911544 SRR5911547 SRR5911548 SRR5911549 SRR5911552 SRR5911554 SRR5911560 SRR5911566 SRR5911571 SRR5911573 SRR5911578 SRR5911581 SRR5911582 SRR5911587 SRR5911590 SRR5911593 SRR5911595 SRR5911597 SRR5911602 SRR5911605 SRR5911606 SRR5911607 SRR5911608 SRR5911621 SRR5911623 SRR5911628 SRR5911636 SRR5911644 SRR5911646 SRR5911649 SRR5911653 SRR5911654 SRR5911660 SRR5911663 SRR5911664 SRR5911670 SRR5911680 SRR5911681 SRR5911695 SRR5911696 SRR5911698 SRR5911705 SRR5911706 SRR5911714 SRR5911718 SRR5911721 SRR5911726 SRR5911729 SRR5911731 SRR5911739 SRR5911749 SRR5911751 SRR5911765 SRR5911770 SRR5911774 SRR5911783 SRR5911784 SRR5911786 SRR5911787 SRR5911788 SRR5911789 SRR5911794 SRR5911797 SRR5911807 SRR5911812 SRR5911819 SRR5911820 SRR5911822 SRR5911824 SRR5911827 SRR5911832 SRR5911833 SRR5911838 SRR5911841 SRR5911844 SRR5911845 SRR5911846 SRR5911849 SRR5911857 SRR5911860 SRR5911861 SRR5911864 SRR5911872 SRR5911876 SRR5911892 SRR5911898 SRR5911899 SRR5911905 SRR5911906 SRR5911908 SRR5911909 SRR5911915 SRR5911916 SRR5911922 SRR5911928 SRR5911929 SRR5911930 SRR5911940 SRR5911941 SRR5911943 SRR5911944 SRR5911946 SRR5911947 SRR5911953 SRR5911955 SRR5911956 SRR5911968 SRR5911971 SRR5911982 SRR5911987 SRR5911988 SRR5911991 SRR5911992 SRR5911993 SRR5911998 SRR5912005 SRR5912006 SRR5912009 SRR5912014 SRR5912015 SRR5912028 SRR5912029 SRR5912032 SRR5912036 SRR5912037 SRR5912039 SRR5912043 SRR5912044 SRR5912053 SRR5912054 SRR5912061 SRR5912065 SRR5912066 SRR5912072 SRR5912073 SRR5912081 SRR5912082 SRR5912083 SRR5912093 SRR5912094 SRR5912104 SRR5912105 SRR5912111 SRR5912116 -o /scratch/rc61809/Kremling_RNA-seq/Zma2_tpm_matrix.txt
#!/bin/bash
#SBATCH --job-name=salmon_quantmerge_kremling2018_counts
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --ntasks-per-node=1
#SBATCH --cpus-per-task=1
#SBATCH --mem=1G
#SBATCH --time=1:00:00
#SBATCH --output=salmon_quantmerge_counts_Zma2.out
#SBATCH --error=salmon_quantmerge_counts_Zma2.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml Salmon/1.9.0-GCC-11.3.0 # Load software module

salmon quantmerge -c numreads --quants SRR5909626_Zma2_quant SRR5909627_Zma2_quant SRR5909633_Zma2_quant SRR5909635_Zma2_quant SRR5909639_Zma2_quant SRR5909642_Zma2_quant SRR5909645_Zma2_quant SRR5909653_Zma2_quant SRR5909655_Zma2_quant SRR5909665_Zma2_quant SRR5909671_Zma2_quant SRR5909672_Zma2_quant SRR5909678_Zma2_quant SRR5909679_Zma2_quant SRR5909687_Zma2_quant SRR5909690_Zma2_quant SRR5909698_Zma2_quant SRR5909699_Zma2_quant SRR5909713_Zma2_quant SRR5909716_Zma2_quant SRR5909721_Zma2_quant SRR5909726_Zma2_quant SRR5909732_Zma2_quant SRR5909739_Zma2_quant SRR5909744_Zma2_quant SRR5909745_Zma2_quant SRR5909752_Zma2_quant SRR5909753_Zma2_quant SRR5909766_Zma2_quant SRR5909774_Zma2_quant SRR5909775_Zma2_quant SRR5909778_Zma2_quant SRR5909782_Zma2_quant SRR5909788_Zma2_quant SRR5909789_Zma2_quant SRR5909792_Zma2_quant SRR5909795_Zma2_quant SRR5909800_Zma2_quant SRR5909806_Zma2_quant SRR5909807_Zma2_quant SRR5909813_Zma2_quant SRR5909814_Zma2_quant SRR5909818_Zma2_quant SRR5909820_Zma2_quant SRR5909823_Zma2_quant SRR5909826_Zma2_quant SRR5909841_Zma2_quant SRR5909861_Zma2_quant SRR5909864_Zma2_quant SRR5909865_Zma2_quant SRR5909866_Zma2_quant SRR5909875_Zma2_quant SRR5909877_Zma2_quant SRR5909882_Zma2_quant SRR5909883_Zma2_quant SRR5909888_Zma2_quant SRR5909891_Zma2_quant SRR5909901_Zma2_quant SRR5909904_Zma2_quant SRR5909907_Zma2_quant SRR5909909_Zma2_quant SRR5909910_Zma2_quant SRR5909911_Zma2_quant SRR5909913_Zma2_quant SRR5909917_Zma2_quant SRR5909918_Zma2_quant SRR5909919_Zma2_quant SRR5909922_Zma2_quant SRR5909930_Zma2_quant SRR5909931_Zma2_quant SRR5909936_Zma2_quant SRR5909939_Zma2_quant SRR5909942_Zma2_quant SRR5909943_Zma2_quant SRR5909948_Zma2_quant SRR5909951_Zma2_quant SRR5909952_Zma2_quant SRR5909959_Zma2_quant SRR5909964_Zma2_quant SRR5909965_Zma2_quant SRR5909972_Zma2_quant SRR5909973_Zma2_quant SRR5909974_Zma2_quant SRR5909997_Zma2_quant SRR5910013_Zma2_quant SRR5910014_Zma2_quant SRR5910015_Zma2_quant SRR5910020_Zma2_quant SRR5910021_Zma2_quant SRR5910037_Zma2_quant SRR5910039_Zma2_quant SRR5910043_Zma2_quant SRR5910044_Zma2_quant SRR5910051_Zma2_quant SRR5910052_Zma2_quant SRR5910054_Zma2_quant SRR5910059_Zma2_quant SRR5910061_Zma2_quant SRR5910062_Zma2_quant SRR5910064_Zma2_quant SRR5910065_Zma2_quant SRR5910066_Zma2_quant SRR5910067_Zma2_quant SRR5910068_Zma2_quant SRR5910069_Zma2_quant SRR5910070_Zma2_quant SRR5910071_Zma2_quant SRR5910072_Zma2_quant SRR5910076_Zma2_quant SRR5910084_Zma2_quant SRR5910087_Zma2_quant SRR5910094_Zma2_quant SRR5910102_Zma2_quant SRR5910103_Zma2_quant SRR5910104_Zma2_quant SRR5910116_Zma2_quant SRR5910117_Zma2_quant SRR5910121_Zma2_quant SRR5910122_Zma2_quant SRR5910658_Zma2_quant SRR5910659_Zma2_quant SRR5910663_Zma2_quant SRR5910664_Zma2_quant SRR5910665_Zma2_quant SRR5910668_Zma2_quant SRR5910676_Zma2_quant SRR5910677_Zma2_quant SRR5910679_Zma2_quant SRR5910688_Zma2_quant SRR5910689_Zma2_quant SRR5910694_Zma2_quant SRR5910695_Zma2_quant SRR5910697_Zma2_quant SRR5910704_Zma2_quant SRR5910705_Zma2_quant SRR5910715_Zma2_quant SRR5910718_Zma2_quant SRR5910721_Zma2_quant SRR5910723_Zma2_quant SRR5910724_Zma2_quant SRR5910730_Zma2_quant SRR5910731_Zma2_quant SRR5910732_Zma2_quant SRR5910735_Zma2_quant SRR5910737_Zma2_quant SRR5910738_Zma2_quant SRR5910739_Zma2_quant SRR5910740_Zma2_quant SRR5910749_Zma2_quant SRR5910757_Zma2_quant SRR5910759_Zma2_quant SRR5910760_Zma2_quant SRR5910766_Zma2_quant SRR5910767_Zma2_quant SRR5910771_Zma2_quant SRR5910772_Zma2_quant SRR5910774_Zma2_quant SRR5910779_Zma2_quant SRR5910783_Zma2_quant SRR5910786_Zma2_quant SRR5910788_Zma2_quant SRR5910791_Zma2_quant SRR5910802_Zma2_quant SRR5910805_Zma2_quant SRR5910806_Zma2_quant SRR5910815_Zma2_quant SRR5910817_Zma2_quant SRR5910822_Zma2_quant SRR5910823_Zma2_quant SRR5910829_Zma2_quant SRR5910830_Zma2_quant SRR5910835_Zma2_quant SRR5910843_Zma2_quant SRR5910851_Zma2_quant SRR5910854_Zma2_quant SRR5910855_Zma2_quant SRR5910862_Zma2_quant SRR5910867_Zma2_quant SRR5910868_Zma2_quant SRR5910877_Zma2_quant SRR5910878_Zma2_quant SRR5910881_Zma2_quant SRR5910882_Zma2_quant SRR5910883_Zma2_quant SRR5910890_Zma2_quant SRR5910895_Zma2_quant SRR5910902_Zma2_quant SRR5910908_Zma2_quant SRR5910909_Zma2_quant SRR5910915_Zma2_quant SRR5910916_Zma2_quant SRR5910925_Zma2_quant SRR5910930_Zma2_quant SRR5910935_Zma2_quant SRR5910936_Zma2_quant SRR5910941_Zma2_quant SRR5910943_Zma2_quant SRR5910949_Zma2_quant SRR5910955_Zma2_quant SRR5910956_Zma2_quant SRR5910967_Zma2_quant SRR5910970_Zma2_quant SRR5910971_Zma2_quant SRR5910972_Zma2_quant SRR5910973_Zma2_quant SRR5910981_Zma2_quant SRR5910982_Zma2_quant SRR5910986_Zma2_quant SRR5910993_Zma2_quant SRR5910996_Zma2_quant SRR5910997_Zma2_quant SRR5911002_Zma2_quant SRR5911004_Zma2_quant SRR5911008_Zma2_quant SRR5911011_Zma2_quant SRR5911014_Zma2_quant SRR5911020_Zma2_quant SRR5911023_Zma2_quant SRR5911032_Zma2_quant SRR5911035_Zma2_quant SRR5911043_Zma2_quant SRR5911044_Zma2_quant SRR5911045_Zma2_quant SRR5911046_Zma2_quant SRR5911051_Zma2_quant SRR5911054_Zma2_quant SRR5911057_Zma2_quant SRR5911063_Zma2_quant SRR5911064_Zma2_quant SRR5911065_Zma2_quant SRR5911068_Zma2_quant SRR5911070_Zma2_quant SRR5911080_Zma2_quant SRR5911081_Zma2_quant SRR5911088_Zma2_quant SRR5911089_Zma2_quant SRR5911093_Zma2_quant SRR5911099_Zma2_quant SRR5911108_Zma2_quant SRR5911111_Zma2_quant SRR5911117_Zma2_quant SRR5911122_Zma2_quant SRR5911125_Zma2_quant SRR5911128_Zma2_quant SRR5911129_Zma2_quant SRR5911136_Zma2_quant SRR5911139_Zma2_quant SRR5911145_Zma2_quant SRR5911146_Zma2_quant SRR5911148_Zma2_quant SRR5911160_Zma2_quant SRR5911163_Zma2_quant SRR5911165_Zma2_quant SRR5911172_Zma2_quant SRR5911173_Zma2_quant SRR5911182_Zma2_quant SRR5911184_Zma2_quant SRR5911185_Zma2_quant SRR5911198_Zma2_quant SRR5911203_Zma2_quant SRR5911212_Zma2_quant SRR5911214_Zma2_quant SRR5911219_Zma2_quant SRR5911220_Zma2_quant SRR5911222_Zma2_quant SRR5911227_Zma2_quant SRR5911229_Zma2_quant SRR5911231_Zma2_quant SRR5911236_Zma2_quant SRR5911237_Zma2_quant SRR5911247_Zma2_quant SRR5911248_Zma2_quant SRR5911251_Zma2_quant SRR5911258_Zma2_quant SRR5911259_Zma2_quant SRR5911265_Zma2_quant SRR5911266_Zma2_quant SRR5911277_Zma2_quant SRR5911278_Zma2_quant SRR5911284_Zma2_quant SRR5911290_Zma2_quant SRR5911291_Zma2_quant SRR5911298_Zma2_quant SRR5911302_Zma2_quant SRR5911309_Zma2_quant SRR5911310_Zma2_quant SRR5911312_Zma2_quant SRR5911326_Zma2_quant SRR5911327_Zma2_quant SRR5911329_Zma2_quant SRR5911330_Zma2_quant SRR5911333_Zma2_quant SRR5911337_Zma2_quant SRR5911344_Zma2_quant SRR5911345_Zma2_quant SRR5911347_Zma2_quant SRR5911350_Zma2_quant SRR5911357_Zma2_quant SRR5911360_Zma2_quant SRR5911363_Zma2_quant SRR5911370_Zma2_quant SRR5911375_Zma2_quant SRR5911376_Zma2_quant SRR5911392_Zma2_quant SRR5911393_Zma2_quant SRR5911396_Zma2_quant SRR5911398_Zma2_quant SRR5911399_Zma2_quant SRR5911421_Zma2_quant SRR5911426_Zma2_quant SRR5911427_Zma2_quant SRR5911433_Zma2_quant SRR5911436_Zma2_quant SRR5911437_Zma2_quant SRR5911443_Zma2_quant SRR5911444_Zma2_quant SRR5911448_Zma2_quant SRR5911452_Zma2_quant SRR5911458_Zma2_quant SRR5911459_Zma2_quant SRR5911464_Zma2_quant SRR5911469_Zma2_quant SRR5911472_Zma2_quant SRR5911475_Zma2_quant SRR5911477_Zma2_quant SRR5911479_Zma2_quant SRR5911490_Zma2_quant SRR5911491_Zma2_quant SRR5911494_Zma2_quant SRR5911497_Zma2_quant SRR5911502_Zma2_quant SRR5911504_Zma2_quant SRR5911512_Zma2_quant SRR5911518_Zma2_quant SRR5911519_Zma2_quant SRR5911524_Zma2_quant SRR5911532_Zma2_quant SRR5911534_Zma2_quant SRR5911535_Zma2_quant SRR5911537_Zma2_quant SRR5911538_Zma2_quant SRR5911542_Zma2_quant SRR5911544_Zma2_quant SRR5911547_Zma2_quant SRR5911548_Zma2_quant SRR5911549_Zma2_quant SRR5911552_Zma2_quant SRR5911554_Zma2_quant SRR5911560_Zma2_quant SRR5911566_Zma2_quant SRR5911571_Zma2_quant SRR5911573_Zma2_quant SRR5911578_Zma2_quant SRR5911581_Zma2_quant SRR5911582_Zma2_quant SRR5911587_Zma2_quant SRR5911590_Zma2_quant SRR5911593_Zma2_quant SRR5911595_Zma2_quant SRR5911597_Zma2_quant SRR5911602_Zma2_quant SRR5911605_Zma2_quant SRR5911606_Zma2_quant SRR5911607_Zma2_quant SRR5911608_Zma2_quant SRR5911621_Zma2_quant SRR5911623_Zma2_quant SRR5911628_Zma2_quant SRR5911636_Zma2_quant SRR5911644_Zma2_quant SRR5911646_Zma2_quant SRR5911649_Zma2_quant SRR5911653_Zma2_quant SRR5911654_Zma2_quant SRR5911660_Zma2_quant SRR5911663_Zma2_quant SRR5911664_Zma2_quant SRR5911670_Zma2_quant SRR5911680_Zma2_quant SRR5911681_Zma2_quant SRR5911695_Zma2_quant SRR5911696_Zma2_quant SRR5911698_Zma2_quant SRR5911705_Zma2_quant SRR5911706_Zma2_quant SRR5911714_Zma2_quant SRR5911718_Zma2_quant SRR5911721_Zma2_quant SRR5911726_Zma2_quant SRR5911729_Zma2_quant SRR5911731_Zma2_quant SRR5911739_Zma2_quant SRR5911749_Zma2_quant SRR5911751_Zma2_quant SRR5911765_Zma2_quant SRR5911770_Zma2_quant SRR5911774_Zma2_quant SRR5911783_Zma2_quant SRR5911784_Zma2_quant SRR5911786_Zma2_quant SRR5911787_Zma2_quant SRR5911788_Zma2_quant SRR5911789_Zma2_quant SRR5911794_Zma2_quant SRR5911797_Zma2_quant SRR5911807_Zma2_quant SRR5911812_Zma2_quant SRR5911819_Zma2_quant SRR5911820_Zma2_quant SRR5911822_Zma2_quant SRR5911824_Zma2_quant SRR5911827_Zma2_quant SRR5911832_Zma2_quant SRR5911833_Zma2_quant SRR5911838_Zma2_quant SRR5911841_Zma2_quant SRR5911844_Zma2_quant SRR5911845_Zma2_quant SRR5911846_Zma2_quant SRR5911849_Zma2_quant SRR5911857_Zma2_quant SRR5911860_Zma2_quant SRR5911861_Zma2_quant SRR5911864_Zma2_quant SRR5911872_Zma2_quant SRR5911876_Zma2_quant SRR5911892_Zma2_quant SRR5911898_Zma2_quant SRR5911899_Zma2_quant SRR5911905_Zma2_quant SRR5911906_Zma2_quant SRR5911908_Zma2_quant SRR5911909_Zma2_quant SRR5911915_Zma2_quant SRR5911916_Zma2_quant SRR5911922_Zma2_quant SRR5911928_Zma2_quant SRR5911929_Zma2_quant SRR5911930_Zma2_quant SRR5911940_Zma2_quant SRR5911941_Zma2_quant SRR5911943_Zma2_quant SRR5911944_Zma2_quant SRR5911946_Zma2_quant SRR5911947_Zma2_quant SRR5911953_Zma2_quant SRR5911955_Zma2_quant SRR5911956_Zma2_quant SRR5911968_Zma2_quant SRR5911971_Zma2_quant SRR5911982_Zma2_quant SRR5911987_Zma2_quant SRR5911988_Zma2_quant SRR5911991_Zma2_quant SRR5911992_Zma2_quant SRR5911993_Zma2_quant SRR5911998_Zma2_quant SRR5912005_Zma2_quant SRR5912006_Zma2_quant SRR5912009_Zma2_quant SRR5912014_Zma2_quant SRR5912015_Zma2_quant SRR5912028_Zma2_quant SRR5912029_Zma2_quant SRR5912032_Zma2_quant SRR5912036_Zma2_quant SRR5912037_Zma2_quant SRR5912039_Zma2_quant SRR5912043_Zma2_quant SRR5912044_Zma2_quant SRR5912053_Zma2_quant SRR5912054_Zma2_quant SRR5912061_Zma2_quant SRR5912065_Zma2_quant SRR5912066_Zma2_quant SRR5912072_Zma2_quant SRR5912073_Zma2_quant SRR5912081_Zma2_quant SRR5912082_Zma2_quant SRR5912083_Zma2_quant SRR5912093_Zma2_quant SRR5912094_Zma2_quant SRR5912104_Zma2_quant SRR5912105_Zma2_quant SRR5912111_Zma2_quant SRR5912116_Zma2_quant --names SRR5909626 SRR5909627 SRR5909633 SRR5909635 SRR5909639 SRR5909642 SRR5909645 SRR5909653 SRR5909655 SRR5909665 SRR5909671 SRR5909672 SRR5909678 SRR5909679 SRR5909687 SRR5909690 SRR5909698 SRR5909699 SRR5909713 SRR5909716 SRR5909721 SRR5909726 SRR5909732 SRR5909739 SRR5909744 SRR5909745 SRR5909752 SRR5909753 SRR5909766 SRR5909774 SRR5909775 SRR5909778 SRR5909782 SRR5909788 SRR5909789 SRR5909792 SRR5909795 SRR5909800 SRR5909806 SRR5909807 SRR5909813 SRR5909814 SRR5909818 SRR5909820 SRR5909823 SRR5909826 SRR5909841 SRR5909861 SRR5909864 SRR5909865 SRR5909866 SRR5909875 SRR5909877 SRR5909882 SRR5909883 SRR5909888 SRR5909891 SRR5909901 SRR5909904 SRR5909907 SRR5909909 SRR5909910 SRR5909911 SRR5909913 SRR5909917 SRR5909918 SRR5909919 SRR5909922 SRR5909930 SRR5909931 SRR5909936 SRR5909939 SRR5909942 SRR5909943 SRR5909948 SRR5909951 SRR5909952 SRR5909959 SRR5909964 SRR5909965 SRR5909972 SRR5909973 SRR5909974 SRR5909997 SRR5910013 SRR5910014 SRR5910015 SRR5910020 SRR5910021 SRR5910037 SRR5910039 SRR5910043 SRR5910044 SRR5910051 SRR5910052 SRR5910054 SRR5910059 SRR5910061 SRR5910062 SRR5910064 SRR5910065 SRR5910066 SRR5910067 SRR5910068 SRR5910069 SRR5910070 SRR5910071 SRR5910072 SRR5910076 SRR5910084 SRR5910087 SRR5910094 SRR5910102 SRR5910103 SRR5910104 SRR5910116 SRR5910117 SRR5910121 SRR5910122 SRR5910658 SRR5910659 SRR5910663 SRR5910664 SRR5910665 SRR5910668 SRR5910676 SRR5910677 SRR5910679 SRR5910688 SRR5910689 SRR5910694 SRR5910695 SRR5910697 SRR5910704 SRR5910705 SRR5910715 SRR5910718 SRR5910721 SRR5910723 SRR5910724 SRR5910730 SRR5910731 SRR5910732 SRR5910735 SRR5910737 SRR5910738 SRR5910739 SRR5910740 SRR5910749 SRR5910757 SRR5910759 SRR5910760 SRR5910766 SRR5910767 SRR5910771 SRR5910772 SRR5910774 SRR5910779 SRR5910783 SRR5910786 SRR5910788 SRR5910791 SRR5910802 SRR5910805 SRR5910806 SRR5910815 SRR5910817 SRR5910822 SRR5910823 SRR5910829 SRR5910830 SRR5910835 SRR5910843 SRR5910851 SRR5910854 SRR5910855 SRR5910862 SRR5910867 SRR5910868 SRR5910877 SRR5910878 SRR5910881 SRR5910882 SRR5910883 SRR5910890 SRR5910895 SRR5910902 SRR5910908 SRR5910909 SRR5910915 SRR5910916 SRR5910925 SRR5910930 SRR5910935 SRR5910936 SRR5910941 SRR5910943 SRR5910949 SRR5910955 SRR5910956 SRR5910967 SRR5910970 SRR5910971 SRR5910972 SRR5910973 SRR5910981 SRR5910982 SRR5910986 SRR5910993 SRR5910996 SRR5910997 SRR5911002 SRR5911004 SRR5911008 SRR5911011 SRR5911014 SRR5911020 SRR5911023 SRR5911032 SRR5911035 SRR5911043 SRR5911044 SRR5911045 SRR5911046 SRR5911051 SRR5911054 SRR5911057 SRR5911063 SRR5911064 SRR5911065 SRR5911068 SRR5911070 SRR5911080 SRR5911081 SRR5911088 SRR5911089 SRR5911093 SRR5911099 SRR5911108 SRR5911111 SRR5911117 SRR5911122 SRR5911125 SRR5911128 SRR5911129 SRR5911136 SRR5911139 SRR5911145 SRR5911146 SRR5911148 SRR5911160 SRR5911163 SRR5911165 SRR5911172 SRR5911173 SRR5911182 SRR5911184 SRR5911185 SRR5911198 SRR5911203 SRR5911212 SRR5911214 SRR5911219 SRR5911220 SRR5911222 SRR5911227 SRR5911229 SRR5911231 SRR5911236 SRR5911237 SRR5911247 SRR5911248 SRR5911251 SRR5911258 SRR5911259 SRR5911265 SRR5911266 SRR5911277 SRR5911278 SRR5911284 SRR5911290 SRR5911291 SRR5911298 SRR5911302 SRR5911309 SRR5911310 SRR5911312 SRR5911326 SRR5911327 SRR5911329 SRR5911330 SRR5911333 SRR5911337 SRR5911344 SRR5911345 SRR5911347 SRR5911350 SRR5911357 SRR5911360 SRR5911363 SRR5911370 SRR5911375 SRR5911376 SRR5911392 SRR5911393 SRR5911396 SRR5911398 SRR5911399 SRR5911421 SRR5911426 SRR5911427 SRR5911433 SRR5911436 SRR5911437 SRR5911443 SRR5911444 SRR5911448 SRR5911452 SRR5911458 SRR5911459 SRR5911464 SRR5911469 SRR5911472 SRR5911475 SRR5911477 SRR5911479 SRR5911490 SRR5911491 SRR5911494 SRR5911497 SRR5911502 SRR5911504 SRR5911512 SRR5911518 SRR5911519 SRR5911524 SRR5911532 SRR5911534 SRR5911535 SRR5911537 SRR5911538 SRR5911542 SRR5911544 SRR5911547 SRR5911548 SRR5911549 SRR5911552 SRR5911554 SRR5911560 SRR5911566 SRR5911571 SRR5911573 SRR5911578 SRR5911581 SRR5911582 SRR5911587 SRR5911590 SRR5911593 SRR5911595 SRR5911597 SRR5911602 SRR5911605 SRR5911606 SRR5911607 SRR5911608 SRR5911621 SRR5911623 SRR5911628 SRR5911636 SRR5911644 SRR5911646 SRR5911649 SRR5911653 SRR5911654 SRR5911660 SRR5911663 SRR5911664 SRR5911670 SRR5911680 SRR5911681 SRR5911695 SRR5911696 SRR5911698 SRR5911705 SRR5911706 SRR5911714 SRR5911718 SRR5911721 SRR5911726 SRR5911729 SRR5911731 SRR5911739 SRR5911749 SRR5911751 SRR5911765 SRR5911770 SRR5911774 SRR5911783 SRR5911784 SRR5911786 SRR5911787 SRR5911788 SRR5911789 SRR5911794 SRR5911797 SRR5911807 SRR5911812 SRR5911819 SRR5911820 SRR5911822 SRR5911824 SRR5911827 SRR5911832 SRR5911833 SRR5911838 SRR5911841 SRR5911844 SRR5911845 SRR5911846 SRR5911849 SRR5911857 SRR5911860 SRR5911861 SRR5911864 SRR5911872 SRR5911876 SRR5911892 SRR5911898 SRR5911899 SRR5911905 SRR5911906 SRR5911908 SRR5911909 SRR5911915 SRR5911916 SRR5911922 SRR5911928 SRR5911929 SRR5911930 SRR5911940 SRR5911941 SRR5911943 SRR5911944 SRR5911946 SRR5911947 SRR5911953 SRR5911955 SRR5911956 SRR5911968 SRR5911971 SRR5911982 SRR5911987 SRR5911988 SRR5911991 SRR5911992 SRR5911993 SRR5911998 SRR5912005 SRR5912006 SRR5912009 SRR5912014 SRR5912015 SRR5912028 SRR5912029 SRR5912032 SRR5912036 SRR5912037 SRR5912039 SRR5912043 SRR5912044 SRR5912053 SRR5912054 SRR5912061 SRR5912065 SRR5912066 SRR5912072 SRR5912073 SRR5912081 SRR5912082 SRR5912083 SRR5912093 SRR5912094 SRR5912104 SRR5912105 SRR5912111 SRR5912116 -o Zma2_counts_matrix.txt

RNAseq - additional Sanity check

Additional analyses were comprised to confirm that low rRNA was present in samples (SortMErna) and that there was low contamination rate across samples (Kraken2). Given that all genotypes were mapped against the B73 predicted transcriptome and that there is high genetic variability across maize genotypes, a relatively low mapping rate would be expected even with low bacterial or rRNA contamination. In the future, we plan to employ the maize pan-transcriptome in further analyses.

16S - generation of the ASVs

Creating Qiime2 artifact

First step before any further analyses is to import Cutadapt reads into a Qiime2 artifact (using QIIME2 v2023.7 on Sapelo2):

#!/bin/bash
#SBATCH --job-name=import_artifact_wallace2018
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --ntasks-per-node=1
#SBATCH --cpus-per-task=1
#SBATCH --mem=1G
#SBATCH --time=2:00:00
#SBATCH --output=import_artifact_assingle_fw_q20_150bp.out
#SBATCH --error=import_artifact_assingle_fw_q20_150bp.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml QIIME2/2023.7 # Load software module

qiime tools import \
  --type 'SampleData[SequencesWithQuality]' \
  --input-path /scratch/rc61809/16S_pipeline/import_reads_qiime2_artifact/manifest_file_assingle_forward_q20_150bp.tsv\
  --output-path paired-end_wallace2018_assingle_forward_q20_150bp.qza \
  --input-format SingleEndFastqManifestPhred33V2

Manifest file:

Creating artifacts for raw reads:

#!/bin/bash
#SBATCH --job-name=import_artifact_rawreads_wallace2018
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=1
#SBATCH --ntasks-per-node=1
#SBATCH --cpus-per-task=1
#SBATCH --mem=1G
#SBATCH --time=10:00:00
#SBATCH --output=import_rawreads_artifact.out
#SBATCH --error=import_rawreads_artifact.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml QIIME2/2023.7 # Load software module

qiime tools import \
  --type 'SampleData[SequencesWithQuality]' \
  --input-path /scratch/rc61809/16S_pipeline/import_reads_qiime2_artifact/manifest_file_rawreads.tsv \
  --output-path single-end_wallace2018.qza \
  --input-format SingleEndFastqManifestPhred33V2

Manifest file:

Generating ASVs with Dada2

Amplicon sequence variants were generated with dada2 denoise-single, considering only forward reads that passed Cutadapt criteria (minimum quality of 20 in both sides, reads still having at least 150bp after trimming):

#!/bin/bash
#SBATCH --job-name=dada2_qiime2_wallace2018
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=16
#SBATCH --ntasks-per-node=16
#SBATCH --cpus-per-task=2
#SBATCH --mem=44G
#SBATCH --time=10:00:00
#SBATCH --output=dada2_qiime2_as_single_q20_150bp.out
#SBATCH --error=dada2_qiime2_as_single_q20_150bp.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml QIIME2/2023.7  # Load software module

qiime dada2 denoise-single --i-demultiplexed-seqs /scratch/rc61809/16S_pipeline/import_reads_qiime2_artifact/paired-end_wallace2018_assingle_forward_q20_150bp.qza --p-trunc-len 0 --p-n-threads 16 --o-table table-paired-end_wallace2018_assingle_forward_q20_150bp-dada2.qza --o-representative-sequences rep-seqs-paired-end_wallace2018_assingle_forward_q20_150bp-dada2.qza --o-denoising-stats stats-paired-end_wallace2018_assingle_forward_q20_150bp-dada2.qza

Key highlights on poster:

  • Short ASVs comprising most of ASV frequencies in several samples
  • A considerable portion of samples have most of ASVs as 'unassigned' taxon in classification stage

Amplicon sequence variants were also generated with dada2 denoise-single, considering only forward reads, but using the raw reads setting a truncate position of 180bp:

#!/bin/bash
#SBATCH --job-name=dada2_qiime2_wallace2018
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=16
#SBATCH --ntasks-per-node=16
#SBATCH --cpus-per-task=2
#SBATCH --mem=44G
#SBATCH --time=10:00:00
#SBATCH --output=dada2_qiime2_as_single_rawreads.out
#SBATCH --error=dada2_qiime2_as_single_rawreads.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml QIIME2/2023.7
ml q2-dada2/2023.5.0-QIIME2-2023.7

qiime dada2 denoise-single --i-demultiplexed-seqs /scratch/rc61809/16S_pipeline/import_reads_qiime2_artifact/single-end_wallace2018.qza --p-trunc-len 180 --p-n-threads 16 --o-table table-paired-end_wallace2018_assingle_forward_rawreads-dada2.qza --o-representative-sequences rep-seqs-paired-end_wallace2018_assingle_forward_rawreads-dada2.qza --o-denoising-stats stats-paired-end_wallace2018_assingle_forward_rawreads-dada2.qza

Key highlights on poster:

  • A considerable portion of samples have most of ASVs as 'unassigned' taxon in classification stage (similar to Dada2 after cutadapt)

Classifying ASVs (forward reads only)

Pre-trained model

  • silva-138-99-nb-classifier.qza (SILVA 138 Full length)

This is a pre-trained model available in Qiime2 data resources page.

Actual classifier

Running classifier for Forward, for ASVs generated from cutadapt (fw, q20 both sides, min 150bp):

#!/bin/bash
#SBATCH --job-name=feature-classifier_qiime2_wallace2018
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=8
#SBATCH --ntasks-per-node=8
#SBATCH --cpus-per-task=2
#SBATCH --mem=44G
#SBATCH --time=10:00:00
#SBATCH --output=feature_classifier_q20_150bp_qiime2.out
#SBATCH --error=feature_classifier_q20_150bp_qiime2.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml QIIME2/2023.7  # Load software module

qiime feature-classifier classify-sklearn \
  --i-classifier ../silva-138-99-nb-classifier.qza \
  --i-reads /scratch/rc61809/16S_pipeline/dada2/q20_150bp/rep-seqs-paired-end_wallace2018_assingle_forward_q20_150bp-dada2.qza \
  --o-classification silva_taxonomy_fw_q20_150bp.qza

Running classifier for Forward, for ASVs generated from raw reads:

#!/bin/bash
#SBATCH --job-name=feature-classifier_qiime2_wallace2018
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=8
#SBATCH --ntasks-per-node=8
#SBATCH --cpus-per-task=2
#SBATCH --mem=44G
#SBATCH --time=10:00:00
#SBATCH --output=feature_classifier_rawreads_qiime2.out
#SBATCH --error=feature_classifier_rawreads_qiime2.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml QIIME2/2023.7  # Load software module

qiime feature-classifier classify-sklearn \
  --i-classifier ../silva-138-99-nb-classifier.qza \
  --i-reads /scratch/rc61809/16S_pipeline/dada2/rawreads/rep-seqs-paired-end_wallace2018_assingle_forward_rawreads-dada2.qza \
  --o-classification silva_taxonomy_fw_rawreads.qza

Generating taxonomic bar plots

Taxonomic bar plots were generated with the qiime taxa barplot tool (dada2 after cutadapt):

#!/bin/bash
#SBATCH --job-name=taxonomic_bar_plot_qiime2_wallace2018
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=8
#SBATCH --ntasks-per-node=8
#SBATCH --cpus-per-task=2
#SBATCH --mem=2G
#SBATCH --time=2:00:00
#SBATCH --output=taxonomic_bar_plot_q20_150bp_qiime2.out
#SBATCH --error=taxonomic_bar_plot_q20_150bp_qiime2.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml QIIME2/2023.7  # Load software module

qiime taxa barplot --i-table ../../dada2/q20_150bp/table-paired-end_wallace2018_assingle_forward_q20_150bp-dada2.qza --i-taxonomy silva_taxonomy_fw_q20_150bp.qza --m-metadata-file /home/rc61809/16S_Wallace2018/Metadata/sample-metadata.tsv --o-visualization taxa-bar-plots_assingle_forward_q20_150bp_silva.qza

Taxonomic bar plots with qiime taxa barplot tool (dada2 with raw reads):

#!/bin/bash
#SBATCH --job-name=taxonomic_bar_plot_qiime2_wallace2018
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=8
#SBATCH --ntasks-per-node=8
#SBATCH --cpus-per-task=2
#SBATCH --mem=2G
#SBATCH --time=2:00:00
#SBATCH --output=taxonomic_bar_plot_rawreads_qiime2.out
#SBATCH --error=taxonomic_bar_plot_rawreads_qiime2.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml QIIME2/2023.7  # Load software module

qiime taxa barplot --i-table ../../dada2/rawreads/table-paired-end_wallace2018_assingle_forward_rawreads-dada2.qza --i-taxonomy silva_taxonomy_fw_rawreads.qza --m-metadata-file /home/rc61809/16S_Wallace2018/Metadata/sample-metadata.tsv --o-visualization taxa-bar-plots_assingle_forward_rawreads_silva.qza

16S - Kraken2 analyses

Creating Kraken2 database with SILVA sequences

#!/bin/bash
#SBATCH --job-name=build_kraken2db_silva_wallace2018
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=8
#SBATCH --ntasks-per-node=8
#SBATCH --cpus-per-task=2
#SBATCH --mem=30G
#SBATCH --time=1-00:00:00
#SBATCH --output=build_kraken2db_silva_wallace2018.out
#SBATCH --error=build_kraken2db_silva_wallace2018.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

ml Kraken2/2.1.3-gompi-2022a  # Load software module

DBNAME=/scratch/rc61809/16S_pipeline/Kraken2/database

kraken2-build --download-taxonomy --threads 8 --db $DBNAME
kraken2-build --db $DBNAME --special silva
kraken2-build --build --threads 8 --db $DBNAME

Running Kraken2 with the ASV representative sequences

Running Kraken2 with the ASV representative sequences for Dada2 results using raw reads:

#!/bin/bash
#SBATCH --job-name=kraken2_wallace2018_silva
#SBATCH --partition=batch
#SBATCH --nodes=1
#SBATCH --ntasks=16
#SBATCH --ntasks-per-node=16
#SBATCH --cpus-per-task=2
#SBATCH --mem=20G
#SBATCH --time=10:00:00
#SBATCH --output=kraken2_wallace2018_asvs.out
#SBATCH --error=kraken2_wallace2018_asvs.err
#SBATCH [email protected]
#SBATCH --mail-type=ALL

cd $SLURM_SUBMIT_DIR

FASTA=/scratch/rc61809/16S_pipeline/dada2/rawreads/rep-seqs-paired-end_wallace2018_assingle_forward_rawreads-dada2/repseqs_dada2_rawreads_180bp_dna-sequences.fasta
REPORTFILE=repseqs_dada2_rawreads_180bp_kraken2_silva_report.txt
OUTFILE=repseqs_dada2_rawreads_180bp_kraken2_silva.txt
DBNAME=/scratch/rc61809/16S_pipeline/Kraken2/database
OUTDIR=kraken2_out

if [ ! -d $OUTDIR ]; then
  mkdir $OUTDIR
fi

ml Kraken2/2.1.3-gompi-2022a  # Load software module

kraken2 --db $DBNAME --threads 16 --use-names --report $OUTDIR/$REPORTFILE --output $OUTDIR/$OUTFILE $FASTA

Integration of RNAseq and 16S - correlation analyses

OTU count matrix

The OTU table for downstream analyses was obtained from Dr. Wallace FigShare repository associated with the original publication (Wallace et al (2018)).

Prior to any analysis, I generated a feature table from the .biom file with biom in qiime2-amplicon-2023.9:

biom convert -i 2f_otu_table.sample_filtered.no_mitochondria_chloroplast.biom -o 2f_otu_table.sample_filtered.no_mitochondria_chloroplast_taxonomy.tsv --to-tsv --header-key taxonomy

TSV file (includes one column with taxon associated with each OTU):

RNA matrix

Raw tables from salmon quantmerge:

Actual correlation analyses

Here is the notebook PDF with correlation analyses.

Development of CoNekT Grasses Microbiome

Currently, the platform is being developed in a private GitLab repository, which will be publicly available open publication, or in a future presentation of a tested/mature version.

Figure generation

Codes to generate part of the figures are available here.

Laboratories involved:

Institutional support:

  • Center for Nuclear Energy in Agriculture (CENA), University of Sao Paulo (USP), Piracicaba, SP, Brazil
  • University of Georgia (UGA), Athens, GA, USA

Financial support: Sao Paulo Research Foundation (FAPESP)

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