Data used in this course

For this course, we will use public data sets from GEO data set GSE76647.

Exercise:

• Go to the GEO page corresponding to entry GSE76647
• What is the organism the samples come from? Which types of cells? What is the goal of the experiment?
• What information do you get about the sequencing protocol?
• Retrieve the SRA codes corresponding to all samplesfrom the SRA RUN Selector)

Data details

• Data set GSE76647 has 10 Homo sapiens samples from differentiated (5-day differentiation) and undifferentiated primary keratinocytes.
  Some of the samples underwent a knock-down of the FOXC1 gene:

GEO ID	SRA ID	Sample name	Differentiation	Condition
GSM2031982	SRR3091420	5p4_25c	undiff	WT
GSM2031983	SRR3091421	5p4_27c	undiff	WT
GSM2031984	SRR3091422	5p4_28c	diff 5 days	WT
GSM2031985	SRR3091423	5p4_29c	diff 5 days	WT
GSM2031986	SRR3091424	5p4_30c	diff 5 days	WT
GSM2031987	SRR3091425	5p4_31cfoxc1	undiff	WT
GSM2031988	SRR3091426	5p4_32cfoxc1	undiff	WT
GSM2031989	SRR3091427	5p4_33cfoxc1	undiff	WT
GSM2031990	SRR3091428	5p4_34cfoxc1	diff 5 days	WT
GSM2031991	SRR3091429	5p4_35cfoxc1	diff 5 days	WT

• The raw data can be downloaded as follows, using fastq-dump

# Go to the "raw_data" folder
cd ~/rnaseq_course/raw_data

# Download raw data files
for sra in SRR309142{0,1,2,3,4,5,6,7,8,9}
do echo $sra
$RUN fastq-dump --gzip --origfmt --skip-technical --split-files $sra
done

• BACK UP

In case downloading those files is too slow, we have prepared fastq files that correspond to chromosome 6 only. Please download it:

# go to raw data folder
cd ~/rnaseq_course/raw_data

# archive containing the 6 fastq files for chromosome 6 only
wget https://public-docs.crg.es/biocore/projects/training/PHINDaccess2020/fastq_chr6.tar.gz

# extract archive
tar -xvzf fastq_chr6.tar.gz

# remove .tar.gz file
rm fastq_chr6.tar.gz

• How many reads are there per sample?
• What is the read length?

Number of reads:

cd ~/rnaseq_course/raw_data/fastq_chr6

# With grep
zcat SRR3091420_1_chr6.fastq.gz | grep "^@" | wc -l

# With paste
zcat SRR3091420_1_chr6.fastq.gz | paste - - - - | wc -l

# With awk
zcat SRR3091420_1_chr6.fastq.gz | awk 'BEGIN{i=0}{i++;}END{print i/4}'

For all samples:

for fastq in *fastq.gz
do echo $fastq
zcat $fastq | awk 'BEGIN{i=0}{i++;}END{print i/4}'
done

Count read length:

# Count read length for all rows
zcat SRR3091420_1_chr6.fastq.gz | paste - - - - | awk '{print length($2)}'

# Summarize
zcat SRR3091420_1_chr6.fastq.gz | paste - - - - | awk '{print length($2)}' | sort | uniq -c