06-cod-RNAseq-alignment

library(tidyverse)
library(dplyr)
library(magrittr)
library(knitr)
library(ggplot2)
library(plotly)

Code for aligning RNAseq data to reference transcriptome/genome, to be used on Pacific cod RNAseq data.

• Raw reads found here
• Transcriptome downloaded from NCBI, stored here as a part of lab genomic resources

Note: Kallisto pseudoalignment doesn’t necessarily require input reads to be trimmed, provided they are of sufficient quality.

1 Create a Bash variables file

This allows usage of Bash variables (e.g. paths to common directories) across R Markdown chunks.

{
echo "#### Assign Variables ####"
echo ""

echo "# Data directories"
echo 'export cod_dir=/home/shared/8TB_HDD_02/shedurkin/project-cod-temperature'
echo 'export output_dir_top=${cod_dir}/output/06-cod-RNAseq-alignment'
echo 'export raw_fastqc_dir=${cod_dir}/output/05-cod-RNAseq-trimming/raw-fastqc'
echo 'export raw_reads_dir=${cod_dir}/data/05-cod-RNAseq-trimming/raw-reads'
echo 'export trimmed_fastqc_dir=${cod_dir}/output/05-cod-RNAseq-trimming/trimmed-fastqc'
echo 'export trimmed_reads_dir=${cod_dir}/output/05-cod-RNAseq-trimming/trimmed-reads'
echo 'export kallisto_output_dir=${output_dir_top}/kallisto'
echo ""


echo "# Input/Output files"
echo 'export transcriptome_fasta_dir=${cod_dir}/data'
echo 'export transcriptome_fasta_name="GCF_031168955.1_ASM3116895v1_rna"'
echo 'export transcriptome_fasta="${transcriptome_fasta_dir}/${transcriptome_fasta_name}"'
echo 'export kallisto_index_name="G_macrocephalus_kallisto_index.idx"'


echo "# External data URLs and checksums"
echo 'export transcriptome_fasta_url="https://owl.fish.washington.edu/halfshell/genomic-databank/GCF_031168955.1_ASM3116895v1_rna.fna"'
echo 'export transcriptome_checksum="2a6c7c98982727e688f033a9b236725b"'
echo ""


echo "# Paths to programs"
echo 'export kallisto=/home/shared/kallisto/kallisto'
echo 'export multiqc=/home/sam/programs/mambaforge/bin/multiqc'
echo 'export trinity_abund_to_matrix=/home/shared/trinityrnaseq-v2.12.0/util/abundance_estimates_to_matrix.pl'
echo ""


echo "# Set number of CPUs to use"
echo 'export threads=20'
echo ""


echo "# Programs associative array"
echo "declare -A programs_array"
echo "programs_array=("
echo '[kallisto]="${kallisto}" \'
echo '[multiqc]="${multiqc}" \'
echo '[trinity_abund_to_matrix]="${trinity_abund_to_matrix}" \'
echo ")"
} > .bashvars

cat .bashvars

#### Assign Variables ####

# Data directories
export cod_dir=/home/shared/8TB_HDD_02/shedurkin/project-cod-temperature
export output_dir_top=${cod_dir}/output/06-cod-RNAseq-alignment
export raw_fastqc_dir=${cod_dir}/output/05-cod-RNAseq-trimming/raw-fastqc
export raw_reads_dir=${cod_dir}/data/05-cod-RNAseq-trimming/raw-reads
export trimmed_fastqc_dir=${cod_dir}/output/05-cod-RNAseq-trimming/trimmed-fastqc
export trimmed_reads_dir=${cod_dir}/output/05-cod-RNAseq-trimming/trimmed-reads
export kallisto_output_dir=${output_dir_top}/kallisto

# Input/Output files
export transcriptome_fasta_dir=${cod_dir}/data
export transcriptome_fasta_name="GCF_031168955.1_ASM3116895v1_rna"
export transcriptome_fasta="${transcriptome_fasta_dir}/${transcriptome_fasta_name}"
export kallisto_index_name="G_macrocephalus_kallisto_index.idx"
# External data URLs and checksums
export transcriptome_fasta_url="https://owl.fish.washington.edu/halfshell/genomic-databank/GCF_031168955.1_ASM3116895v1_rna.fna"
export transcriptome_checksum="2a6c7c98982727e688f033a9b236725b"

# Paths to programs
export kallisto=/home/shared/kallisto/kallisto
export multiqc=/home/sam/programs/mambaforge/bin/multiqc
export trinity_abund_to_matrix=/home/shared/trinityrnaseq-v2.12.0/util/abundance_estimates_to_matrix.pl

# Set number of CPUs to use
export threads=20

# Programs associative array
declare -A programs_array
programs_array=(
[kallisto]="${kallisto}" \
[multiqc]="${multiqc}" \
[trinity_abund_to_matrix]="${trinity_abund_to_matrix}" \
)

There didn’t seem to be a significant difference in sequence quality following trimming, so for now I’m proceeding with the raw reads (though I’ll likely eventually rerun the kallisto with trimmed)

2 Align to reference transcriptome (Kallisto pseudoalignment)

2.1 Retrieving the reference transcriptome

# Load bash variables into memory
source .bashvars

wget \
--directory-prefix ${transcriptome_fasta_dir} \
--recursive \
--no-check-certificate \
--continue \
--no-host-directories \
--no-directories \
--no-parent \
--quiet \
--execute robots=off \
--accept "${transcriptome_fasta_name}.fna" ${transcriptome_fasta_url}

# Load bash variables into memory
source .bashvars

ls -lh "${transcriptome_fasta_dir}"

total 1.9G
drwxr-xr-x 3 shedurkin labmembers 4.0K Mar  4 11:05 05-cod-RNAseq-trimming
-rw-r--r-- 1 shedurkin labmembers  13K Dec 27 15:45 Cod_RNAseq_NGS_Template_File.xlsx
-rw-r--r-- 1 shedurkin labmembers 2.1K Mar 20 20:55 DESeq2_Sample_Information.csv
-rw-r--r-- 1 shedurkin labmembers  38M Oct 25  2023 Gadus_macrocephalus.coding.gene.V1.cds
-rw-r--r-- 1 shedurkin labmembers 537M Oct 16  2023 GCF_031168955.1_ASM3116895v1_genomic.fna
-rw-r--r-- 1 shedurkin labmembers 351M Oct 16  2023 GCF_031168955.1_ASM3116895v1.gff
-rw-r--r-- 1 shedurkin labmembers 169M Oct 16  2023 GCF_031168955.1_ASM3116895v1_rna.fna
-rw-r--r-- 1 shedurkin labmembers 404M Apr 23 14:29 genomic.gtf
-rw-r--r-- 1 shedurkin labmembers  47K Oct 25  2023 Pcod Temp Growth experiment 2022-23 DATA.xlsx
-rw-r--r-- 1 shedurkin labmembers 231K Mar  4 17:41 Sample.QC.report.of_30-943133806_240118025106.pdf
-rw-r--r-- 1 shedurkin labmembers  12K Mar  4 17:41 Sample.QC.report.of_30-943133806_240118025106.xlsx
-rw-r--r-- 1 shedurkin labmembers  12K Oct 25  2023 temp-experiment.csv
-rw-r--r-- 1 shedurkin labmembers 271M Oct 25  2023 uniprot_sprot_r2023_04.fasta
-rw-r--r-- 1 shedurkin labmembers  88M Apr 17 11:54 uniprot_sprot_r2023_04.fasta.gz

2.2 Verify transcriptome FastA MD5 checksum

# Load bash variables into memory
source .bashvars

cd "${transcriptome_fasta_dir}"

# Checksums file contains other files, so this just looks for the sRNAseq files.
md5sum --check <<< "${transcriptome_checksum}  ${transcriptome_fasta_name}.fna"

GCF_031168955.1_ASM3116895v1_rna.fna: OK

2.3 Building Index

# Load bash variables into memory
source .bashvars

cd "${kallisto_output_dir}"

${programs_array[kallisto]} index \
--threads=${threads} \
--index="${kallisto_index_name}" \
"${transcriptome_fasta}.fna"

# Load bash variables into memory
source .bashvars

ls -lh ${kallisto_output_dir}

total 1.5G
-rw-r--r-- 1 shedurkin labmembers 1.5G Mar 18 16:08 G_macrocephalus_kallisto_index.idx
-rw-r--r-- 1 shedurkin labmembers  20M May  3 15:01 kallisto.isoform.counts.matrix
-rw-r--r-- 1 shedurkin labmembers    0 May  3 15:01 kallisto.isoform.TMM.EXPR.matrix
-rw-r--r-- 1 shedurkin labmembers  24M May  3 15:01 kallisto.isoform.TPM.not_cross_norm
-rw-r--r-- 1 shedurkin labmembers  532 May  3 15:01 kallisto.isoform.TPM.not_cross_norm.runTMM.R
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:46 kallisto_quant_1
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:11 kallisto_quant_10
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:03 kallisto_quant_100
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:04 kallisto_quant_100.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:05 kallisto_quant_107
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:05 kallisto_quant_107.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:07 kallisto_quant_108
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:08 kallisto_quant_108.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:09 kallisto_quant_109
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:10 kallisto_quant_109.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:12 kallisto_quant_10.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:22 kallisto_quant_11
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:13 kallisto_quant_110
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:14 kallisto_quant_110.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:15 kallisto_quant_117
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:16 kallisto_quant_117.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:18 kallisto_quant_118
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:18 kallisto_quant_118.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:20 kallisto_quant_119
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:20 kallisto_quant_119.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:22 kallisto_quant_11.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:33 kallisto_quant_12
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:24 kallisto_quant_120
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:24 kallisto_quant_120.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:26 kallisto_quant_121
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:27 kallisto_quant_121.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:28 kallisto_quant_127
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:29 kallisto_quant_127.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:30 kallisto_quant_128
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:30 kallisto_quant_128.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:31 kallisto_quant_129
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:31 kallisto_quant_129.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:33 kallisto_quant_12.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:39 kallisto_quant_13
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:34 kallisto_quant_131
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:34 kallisto_quant_131.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:35 kallisto_quant_137
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:35 kallisto_quant_137.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:36 kallisto_quant_138
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:36 kallisto_quant_138.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:37 kallisto_quant_139
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:37 kallisto_quant_139.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:39 kallisto_quant_13.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:40 kallisto_quant_140
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:40 kallisto_quant_140.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:41 kallisto_quant_147
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:41 kallisto_quant_147.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:43 kallisto_quant_148
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:44 kallisto_quant_148.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:51 kallisto_quant_149
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:52 kallisto_quant_149.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 22:52 kallisto_quant_150
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 22:53 kallisto_quant_150.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:37 kallisto_quant_18
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:37 kallisto_quant_18.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:41 kallisto_quant_19
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:39 kallisto_quant_19-G
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:40 kallisto_quant_19-G.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:41 kallisto_quant_19.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:43 kallisto_quant_19-S
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:44 kallisto_quant_19-S.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:46 kallisto_quant_1.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:59 kallisto_quant_2
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:50 kallisto_quant_20
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:48 kallisto_quant_20-G
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:49 kallisto_quant_20-G.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:50 kallisto_quant_20.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:52 kallisto_quant_20-S
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:52 kallisto_quant_20-S.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:54 kallisto_quant_21
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:54 kallisto_quant_21.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:56 kallisto_quant_28
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:56 kallisto_quant_28.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:57 kallisto_quant_29
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:58 kallisto_quant_29.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 18:00 kallisto_quant_2.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:53 kallisto_quant_3
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 18:01 kallisto_quant_30
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 18:02 kallisto_quant_30.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 18:03 kallisto_quant_31
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 18:04 kallisto_quant_31.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:47 kallisto_quant_37
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:48 kallisto_quant_37.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:49 kallisto_quant_38
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:49 kallisto_quant_38.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:51 kallisto_quant_39
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:51 kallisto_quant_39.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:53 kallisto_quant_3.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:04 kallisto_quant_4
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:54 kallisto_quant_40
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:55 kallisto_quant_40.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:56 kallisto_quant_41
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:56 kallisto_quant_41.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:58 kallisto_quant_47
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:58 kallisto_quant_47.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:00 kallisto_quant_48
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:00 kallisto_quant_48.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:02 kallisto_quant_49
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:02 kallisto_quant_49.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:04 kallisto_quant_4.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:26 kallisto_quant_5
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:06 kallisto_quant_50
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:06 kallisto_quant_50.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:14 kallisto_quant_57
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:10 kallisto_quant_57-G
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:11 kallisto_quant_57-G.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:14 kallisto_quant_57.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:15 kallisto_quant_57-S
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:15 kallisto_quant_57-S.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:20 kallisto_quant_58
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:18 kallisto_quant_58-G
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:18 kallisto_quant_58-G.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:20 kallisto_quant_58.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:22 kallisto_quant_58-S
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:22 kallisto_quant_58-S.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:24 kallisto_quant_59
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:24 kallisto_quant_59.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:26 kallisto_quant_5.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:28 kallisto_quant_60
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:28 kallisto_quant_60.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:30 kallisto_quant_67
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:30 kallisto_quant_67.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:32 kallisto_quant_68
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:32 kallisto_quant_68.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:34 kallisto_quant_69
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:34 kallisto_quant_69.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:35 kallisto_quant_70
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:36 kallisto_quant_70.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:37 kallisto_quant_78
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:37 kallisto_quant_78.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:39 kallisto_quant_79
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:39 kallisto_quant_79.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:40 kallisto_quant_80
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:41 kallisto_quant_80.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:42 kallisto_quant_83
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:43 kallisto_quant_83.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:44 kallisto_quant_88
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:44 kallisto_quant_88.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:46 kallisto_quant_90
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:46 kallisto_quant_90.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:48 kallisto_quant_91
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:48 kallisto_quant_91.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:50 kallisto_quant_97
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:50 kallisto_quant_97.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:52 kallisto_quant_98
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:52 kallisto_quant_98.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:53 kallisto_quant_99
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:54 kallisto_quant_99.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:55 kallisto_quant_RESUB-116
-rw-r--r-- 1 shedurkin labmembers 5.3K Apr 29 21:55 kallisto_quant_RESUB-116.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:57 kallisto_quant_RESUB-156
-rw-r--r-- 1 shedurkin labmembers 5.3K Apr 29 21:57 kallisto_quant_RESUB-156.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:58 kallisto_quant_RESUB-36
-rw-r--r-- 1 shedurkin labmembers 5.3K Apr 29 21:59 kallisto_quant_RESUB-36.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 22:54 kallisto_quant_RESUB-76
-rw-r--r-- 1 shedurkin labmembers 5.3K Apr 29 22:54 kallisto_quant_RESUB-76.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 22:13 kallisto_quant_RESUB-94
-rw-r--r-- 1 shedurkin labmembers 5.3K Apr 29 22:14 kallisto_quant_RESUB-94.log

2.4 Sample Quantification

Kallisto can run quantification on either single- or paired-end reads. The default option is paired-end, which requires the input of an even number of paired fastq files (e.g., pairA_R1.fastq, pairA_R2.fastq). To use single-end mode, include the –single flag, as well as -l (–fragment-length=DOUBLE, estimated avg. fragment length) and -s (–sd=DOUBLE, estimates stand. dev. of fragment length), and a number of fastq files. Again, gzipped files are acceptable.

Kallisto quant is rather finicky about how you input sets of paired reads, and you can only input a single pair at a time. To circumvent, I’ll create a quantification function and apply it iteratively to each pair of reads using a loop.

# Load bash variables into memory
source .bashvars

# Function to run kallisto quant. Takes two (paired) reads as input, outputs to sample-associated directory
run_kallisto_quant() {
    source .bashvars  # Source .bashvars inside the function to make its variables accessible
    local R1_fastq=${1}
    local R2_fastq=${2}
    
    cd ${kallisto_output_dir}
    sample_num=$(basename "${R1_fastq}" "_R1_001.fastq.gz")
    mkdir kallisto_quant_${sample_num}

    ${programs_array[kallisto]} quant \
        --threads=${threads} \
        --index="${kallisto_output_dir}/${kallisto_index_name}" \
        --output-dir="${kallisto_output_dir}/kallisto_quant_${sample_num}" \
        --bootstrap-samples=100 \
        ${raw_reads_dir}/${R1_fastq} ${raw_reads_dir}/${R2_fastq} \
        &> "${kallisto_output_dir}/kallisto_quant_${sample_num}.log"
}



# Iteratively apply run_kallisto_quant on each pair of input reads
for file_r1 in "${raw_reads_dir}"/*_R1_001.fastq.gz; do
    # Extract the sample name from the file name
    sample_name=$(basename "${file_r1}" "_R1_001.fastq.gz")

    # Form the file names (function takes input file names, not paths)
    file_r1_name="${sample_name}_R1_001.fastq.gz"
    file_r2_name="${sample_name}_R2_001.fastq.gz"

    # Check that the sample hasn't already been quantified
    if [ ! -d "${kallisto_output_dir}/kallisto_quant_${sample_name}" ]; then
    
        # Check if the corresponding R2 file exists
        if [ -e "${raw_reads_dir}/${file_r2}" ]; then
            # Run kallisto quant on the file pair
            run_kallisto_quant "${file_r1_name}" "${file_r2_name}" 

            echo "Processed sample: ${sample_name}"
        fi
    else
        echo "Sample already processed: ${sample_name}"
    fi
done

Check that we have the appropriate number of output folders. We should have one log file for each pair of reads

# Load bash variables into memory
source .bashvars

# Count number of raw read files
cd ${raw_reads_dir}
echo "Number of raw reads:"
ls -1 | wc -l

# Count number of kallisto output 
cd ${kallisto_output_dir}
echo "Number of output log files"
find . -type f -name "*.log" | wc -l

Number of raw reads:
158
Number of output log files
79

2.5 MultiQC on Kallisto output logs

# Load bash variables into memory
source .bashvars

############ RUN MULTIQC ############
echo "Beginning MultiQC on raw FastQC..."
echo ""

${programs_array[multiqc]} ${kallisto_output_dir}/*.log -o ${output_dir_top}

echo ""
echo "MultiQC on raw FastQs complete."
echo ""

############ END MULTIQC ############

echo "Removing FastQC zip files."
echo ""
rm ${output_dir_top}/*.zip
echo "FastQC zip files removed."
echo ""

# View directory contents
ls -lh ${output_dir_top}

I also want to include the treatment/tank info when plotting alignment rates across samples

# Load multiqc stats
kallisto_multiqc <- read.csv("../output/06-cod-RNAseq-alignment/multiqc_data/multiqc_kallisto.txt", sep = '\t')
# Adjust sample name formatting (to prep for join)
kallisto_multiqc$Sample <- gsub("_R1_001", "", kallisto_multiqc$Sample) 
kallisto_multiqc$Sample <- paste("sample_", kallisto_multiqc$Sample, sep = "")
# Load experimental data
cod_sample_info_OG <- read.csv("../data/DESeq2_Sample_Information.csv")

kallisto_multiqc_plustreatment <- left_join(cod_sample_info_OG, kallisto_multiqc, by = c("sample_name" = "Sample")) %>% 
  na.omit()
kallisto_multiqc_plustreatment <- kallisto_multiqc_plustreatment[order(kallisto_multiqc_plustreatment$sample_number),]

ggplot(kallisto_multiqc_plustreatment,
       aes(x=reorder(sample_name, sample_number), y=percent_aligned, fill=as.factor(temp_treatment))) +
  geom_bar(stat="identity") +
  theme(axis.text.x = element_text(angle = 60, vjust = 1, hjust=1, size=7))

ggplot(kallisto_multiqc_plustreatment,
       aes(x=reorder(sample_name, sample_number), y=total_reads, fill=as.factor(temp_treatment))) +
  geom_bar(stat="identity") +
  theme(axis.text.x = element_text(angle = 60, vjust = 1, hjust=1, size=7))

# sample149 is kind of throwing off the visualization, so lets remove and redo
ggplot(kallisto_multiqc_plustreatment[kallisto_multiqc_plustreatment$sample_name != "sample_149", ],
       aes(x=reorder(sample_name, sample_number), y=total_reads, fill=as.factor(temp_treatment))) +
  geom_bar(stat="identity") +
  theme(axis.text.x = element_text(angle = 60, vjust = 1, hjust=1, size=7))

ggplot(kallisto_multiqc_plustreatment,
       aes(x=reorder(sample_name, sample_number), y=fragment_length, fill=as.factor(temp_treatment))) +
  geom_bar(stat="identity") +
  theme(axis.text.x = element_text(angle = 60, vjust = 1, hjust=1, size=7))

2.6 Trinity Matrix with Kallisto Output

# Load bash variables into memory
source .bashvars

cd ${kallisto_output_dir}

${programs_array[trinity_abund_to_matrix]} \
--est_method 'kallisto' \
--gene_trans_map 'none' \
--out_prefix 'kallisto' \
--name_sample_by_basedir ${kallisto_output_dir}/kallisto_quant_*/abundance.tsv

ls -lh ${kallisto_output_dir}

-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_100/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_107/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_108/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_109/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_10/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_110/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_117/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_118/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_119/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_11/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_120/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_121/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_127/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_128/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_129/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_12/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_131/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_137/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_138/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_139/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_13/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_140/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_147/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_148/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_149/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_150/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_18/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_19/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_19-G/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_19-S/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_1/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_20/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_20-G/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_20-S/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_21/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_28/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_29/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_2/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_30/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_31/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_37/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_38/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_39/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_3/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_40/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_41/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_47/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_48/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_49/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_4/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_50/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_57/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_57-G/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_57-S/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_58/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_58-G/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_58-S/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_59/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_5/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_60/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_67/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_68/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_69/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_70/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_78/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_79/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_80/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_83/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_88/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_90/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_91/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_97/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_98/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_99/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_RESUB-116/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_RESUB-156/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_RESUB-36/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_RESUB-76/abundance.tsv
-reading file: /home/shared/8TB_HDD_02/shedurkin/project-cod-temperature/output/06-cod-RNAseq-alignment/kallisto/kallisto_quant_RESUB-94/abundance.tsv


* Outputting combined matrix.

/home/shared/trinityrnaseq-v2.12.0/util/support_scripts/run_TMM_scale_matrix.pl --matrix kallisto.isoform.TPM.not_cross_norm > kallisto.isoform.TMM.EXPR.matrixCMD: R --no-save --no-restore --no-site-file --no-init-file -q < kallisto.isoform.TPM.not_cross_norm.runTMM.R 1>&2 
sh: 1: R: not found
Error, cmd: R --no-save --no-restore --no-site-file --no-init-file -q < kallisto.isoform.TPM.not_cross_norm.runTMM.R 1>&2  died with ret (32512)  at /home/shared/trinityrnaseq-v2.12.0/util/support_scripts/run_TMM_scale_matrix.pl line 105.
Error, CMD: /home/shared/trinityrnaseq-v2.12.0/util/support_scripts/run_TMM_scale_matrix.pl --matrix kallisto.isoform.TPM.not_cross_norm > kallisto.isoform.TMM.EXPR.matrix died with ret 6400 at /home/shared/trinityrnaseq-v2.12.0/util/abundance_estimates_to_matrix.pl line 385.
total 1.5G
-rw-r--r-- 1 shedurkin labmembers 1.5G Mar 18 16:08 G_macrocephalus_kallisto_index.idx
-rw-r--r-- 1 shedurkin labmembers  20M May  3 15:02 kallisto.isoform.counts.matrix
-rw-r--r-- 1 shedurkin labmembers    0 May  3 15:02 kallisto.isoform.TMM.EXPR.matrix
-rw-r--r-- 1 shedurkin labmembers  24M May  3 15:02 kallisto.isoform.TPM.not_cross_norm
-rw-r--r-- 1 shedurkin labmembers  532 May  3 15:02 kallisto.isoform.TPM.not_cross_norm.runTMM.R
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:46 kallisto_quant_1
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:11 kallisto_quant_10
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:03 kallisto_quant_100
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:04 kallisto_quant_100.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:05 kallisto_quant_107
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:05 kallisto_quant_107.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:07 kallisto_quant_108
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:08 kallisto_quant_108.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:09 kallisto_quant_109
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:10 kallisto_quant_109.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:12 kallisto_quant_10.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:22 kallisto_quant_11
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:13 kallisto_quant_110
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:14 kallisto_quant_110.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:15 kallisto_quant_117
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:16 kallisto_quant_117.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:18 kallisto_quant_118
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:18 kallisto_quant_118.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:20 kallisto_quant_119
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:20 kallisto_quant_119.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:22 kallisto_quant_11.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:33 kallisto_quant_12
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:24 kallisto_quant_120
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:24 kallisto_quant_120.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:26 kallisto_quant_121
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:27 kallisto_quant_121.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:28 kallisto_quant_127
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:29 kallisto_quant_127.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:30 kallisto_quant_128
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:30 kallisto_quant_128.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:31 kallisto_quant_129
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:31 kallisto_quant_129.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:33 kallisto_quant_12.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:39 kallisto_quant_13
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:34 kallisto_quant_131
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:34 kallisto_quant_131.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:35 kallisto_quant_137
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:35 kallisto_quant_137.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:36 kallisto_quant_138
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:36 kallisto_quant_138.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:37 kallisto_quant_139
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:37 kallisto_quant_139.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:39 kallisto_quant_13.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:40 kallisto_quant_140
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:40 kallisto_quant_140.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:41 kallisto_quant_147
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:41 kallisto_quant_147.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:43 kallisto_quant_148
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:44 kallisto_quant_148.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 15:51 kallisto_quant_149
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 15:52 kallisto_quant_149.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 22:52 kallisto_quant_150
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 22:53 kallisto_quant_150.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:37 kallisto_quant_18
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:37 kallisto_quant_18.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:41 kallisto_quant_19
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:39 kallisto_quant_19-G
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:40 kallisto_quant_19-G.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:41 kallisto_quant_19.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:43 kallisto_quant_19-S
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:44 kallisto_quant_19-S.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:46 kallisto_quant_1.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:59 kallisto_quant_2
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:50 kallisto_quant_20
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:48 kallisto_quant_20-G
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:49 kallisto_quant_20-G.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:50 kallisto_quant_20.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:52 kallisto_quant_20-S
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:52 kallisto_quant_20-S.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:54 kallisto_quant_21
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:54 kallisto_quant_21.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:56 kallisto_quant_28
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:56 kallisto_quant_28.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 17:57 kallisto_quant_29
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 17:58 kallisto_quant_29.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 18:00 kallisto_quant_2.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:53 kallisto_quant_3
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 18:01 kallisto_quant_30
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 18:02 kallisto_quant_30.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 18:03 kallisto_quant_31
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 18:04 kallisto_quant_31.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:47 kallisto_quant_37
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:48 kallisto_quant_37.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:49 kallisto_quant_38
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:49 kallisto_quant_38.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:51 kallisto_quant_39
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:51 kallisto_quant_39.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:53 kallisto_quant_3.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:04 kallisto_quant_4
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:54 kallisto_quant_40
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:55 kallisto_quant_40.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:56 kallisto_quant_41
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:56 kallisto_quant_41.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 20:58 kallisto_quant_47
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 20:58 kallisto_quant_47.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:00 kallisto_quant_48
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:00 kallisto_quant_48.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:02 kallisto_quant_49
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:02 kallisto_quant_49.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:04 kallisto_quant_4.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:26 kallisto_quant_5
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:06 kallisto_quant_50
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:06 kallisto_quant_50.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:14 kallisto_quant_57
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:10 kallisto_quant_57-G
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:11 kallisto_quant_57-G.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:14 kallisto_quant_57.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:15 kallisto_quant_57-S
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:15 kallisto_quant_57-S.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:20 kallisto_quant_58
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:18 kallisto_quant_58-G
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:18 kallisto_quant_58-G.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:20 kallisto_quant_58.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:22 kallisto_quant_58-S
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:22 kallisto_quant_58-S.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:24 kallisto_quant_59
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:24 kallisto_quant_59.log
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:26 kallisto_quant_5.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:28 kallisto_quant_60
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:28 kallisto_quant_60.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:30 kallisto_quant_67
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:30 kallisto_quant_67.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:32 kallisto_quant_68
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:32 kallisto_quant_68.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:34 kallisto_quant_69
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:34 kallisto_quant_69.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:35 kallisto_quant_70
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:36 kallisto_quant_70.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:37 kallisto_quant_78
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:37 kallisto_quant_78.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:39 kallisto_quant_79
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:39 kallisto_quant_79.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:40 kallisto_quant_80
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:41 kallisto_quant_80.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:42 kallisto_quant_83
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:43 kallisto_quant_83.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:44 kallisto_quant_88
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:44 kallisto_quant_88.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:46 kallisto_quant_90
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:46 kallisto_quant_90.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:48 kallisto_quant_91
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:48 kallisto_quant_91.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:50 kallisto_quant_97
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:50 kallisto_quant_97.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:52 kallisto_quant_98
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:52 kallisto_quant_98.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:53 kallisto_quant_99
-rw-r--r-- 1 shedurkin labmembers 5.2K Apr 29 21:54 kallisto_quant_99.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:55 kallisto_quant_RESUB-116
-rw-r--r-- 1 shedurkin labmembers 5.3K Apr 29 21:55 kallisto_quant_RESUB-116.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:57 kallisto_quant_RESUB-156
-rw-r--r-- 1 shedurkin labmembers 5.3K Apr 29 21:57 kallisto_quant_RESUB-156.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 21:58 kallisto_quant_RESUB-36
-rw-r--r-- 1 shedurkin labmembers 5.3K Apr 29 21:59 kallisto_quant_RESUB-36.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 22:54 kallisto_quant_RESUB-76
-rw-r--r-- 1 shedurkin labmembers 5.3K Apr 29 22:54 kallisto_quant_RESUB-76.log
drwxr-xr-x 2 shedurkin labmembers 4.0K Apr 29 22:13 kallisto_quant_RESUB-94
-rw-r--r-- 1 shedurkin labmembers 5.3K Apr 29 22:14 kallisto_quant_RESUB-94.log