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@HWI-ST1097:104:D13TNACXX:4:1101:1715:2142 1:N:0:CGATGT
GCGTTGGTGGCATAGTGGTGAGCATAGCTGCCTTCCAAGCAGTTATGGGAG
+
=<@BDDD=A;+2C9F<CB?;CGGA<<ACEE*1?C:D>DE=FC*0BAG?DB6

head /corral-repl/utexas/BioITeam/ngs_course/intro_to_mapping/data/SRR030257_1.fastq

Executing the command above reports that the 2nd sequence has ID = @SRR030257.2 HWI-EAS_4_PE-FC20GCB:6:1:407:767/1, and the sequence TAAGCCAGTCGCCATGGAATATCTGCTTTATTTAGC

wc -l $BI/ngs_course/intro_to_mapping/data/SRR030257_1.fastq

gunzip -c $BI/web/yeast_stuff/Sample_Yeast_L005_R1.cat.fastq.gz | wc -l

The bash shell has a really strange syntax for arithmetic: it uses a double-parenthesis operator. Go figure.

echo $((2368720 / 4))

# setup
cds
mkdir fastqc_test
cd fastqc_test
cp $BI/web/yeast_stuff/Sample_Yeast_L005_R1.cat.fastq.gz .

# running the program
$BI/bin/FastQC/fastqc Sample_Yeast_L005_R1.cat.fastq.gz

drwxrwxr-x 4 abattenh G-803889     4096 May 20 22:59 Sample_Yeast_L005_R1.cat_fastqc
-rw-rw-r-- 1 abattenh G-803889   198239 May 20 22:59 Sample_Yeast_L005_R1.cat_fastqc.zip
-rwxr-xr-x 1 abattenh G-803889 51065629 May 20 22:59 Sample_Yeast_L005_R1.cat.fastq.gz

The Sample_Yeast_L005_R1.cat.fastq.gz file is what we analyzed, so FastQC created the other two items. Sample_Yeast_L005_R1.cat_fastqc is a directory (the "d" in "drwxrwxr-x"), so use ls Sample_Yeast_L005_R1.cat_fastqc to see what's in it. Sample_Yeast_L005_R1.cat_fastqc.zip is just a Zipped (compressed) version of the whole directory.

http://loving.corral.tacc.utexas.edu/bioiteam/yeast_stuff/Sample_Yeast_L005_R1.cat_fastqc/fastqc_report.html

# setup
cds
mkdir samstat_test
cd samstat_test
cp $BI/ngs_course/intro_to_mapping/data/SRR030257_1.fastq .

# run the program
$BI/bin/samstat SRR030257_1.fastq

http://loving.corral.tacc.utexas.edu/bioiteam/SRR030257_1.fastq.html

# setup
cds
mkdir samstat_test2
cd samstat_test2
cp $BI/web/yeast_stuff/yeast_chip_sort.bam .

# run the program
$BI/bin/samstat yeast_chip_sort.bam

http://loving.corral.tacc.utexas.edu/bioiteam/yeast_stuff/yeast_chip_sort.bam.html

module spider fastx_toolkit
module load fastx_toolkit

gunzip -c $BI/web/yeast_stuff/Sample_Yeast_L005_R1.cat.fastq.gz | fastx_trimmer -l 50 -Q 33 > trimmed.fq

You could supply the -z option like this:

gunzip -c $BI/web/yeast_stuff/Sample_Yeast_L005_R1.cat.fastq.gz | fastx_trimmer -l 50 -Q 33 -z > trimmed.fq.gz

Or you could gzip the output yourself:

gunzip -c $BI/web/yeast_stuff/Sample_Yeast_L005_R1.cat.fastq.gz | fastx_trimmer -l 50 -Q 33 | gzip > trimmed.fq.gz

Type fastx_ then tab to see their names
See all the programs like this:

ls $TACC_FASTX_BIN

cutadapt -m 22 -O 10 -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

cutadapt -m 22 -O 10 -a TGATCGTCGGACTGTAGAACTCTGAACGTGTAGA

Please refer to https://wikis.utexas.edu/display/GSAF/Illumina+-+all+flavors for Illumina library adapter layout.

The top strand, 5' to 3', of a read sequence looks like this.

<P5 capture> <indexRead2> <Read 1 primer> [insert] <Read 2 primer> <indexRead1> <P7 capture>

The -a argument to cutadapt is documented as the "sequence of adapter that was ligated to the 3' end". So we care about the <Read 2 primer> for R1 reads, and the <Read 1 primer> for R2 reads.

The "contaminent" for adapter trimming will be the <Read 2 primer> for R1 reads. There is only one Read 2 primer:

AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

The "contaminent" for adapter trimming will be the <Read 1 primer> for R2 reads. However, there are three different Read 1 primers, depending on library construction:

TCTACACGTTCAGAGTTCTACAGTCCGACGATCA    # small RNA sequencing primer site
CAGGTTCAGAGTTCTACAGTCCGACGATCA        # "other"
TCTACACTCTTTCCCTACACGACGCTCTTCCGATCT  # TruSeq Read 1 primer site. This is the RC of the R2 adapter

Since R2 reads are the reverse complement of R1 reads, the R2 adapter contaminent will be the RC of the Read 1 primer used.

For ChIP-seq libraries where reads come from both DNA strands, the TruSeq Read 1 primer is always used.
Since it is the RC of the Read 2 primer, its RC is just the Read 1 primer back
Therefore, for ChIP-seq libraries only one cutadapt command is needed:

raries, both R1 and R2 reads}
cutadapt -a GATCGGAAGAGCACACGTCTGAACTCCAGTCAC

For RNAseq libraries, we use the small RNA sequencing primer as the Read 1 primer.
The contaminent is then the RC of this, minus the 1st and last bases:

TCTACACGTTCAGAGTTCTACAGTCCGACGATCA    # R1 primer - small RNA sequencing Read 1 primer site, 5' to 3'
TGATCGTCGGACTGTAGAACTCTGAACGTGTAGA    # R2 adapter contaminent (RC of R1 small RNA sequencing Read 1 primer)

flexbar -n 1 --adapters adaptors.fna --source example.fastq --target example.ar --format fastq-sanger --adapter-threshold 2 --adapter-min-overlap 6 --adapter-trim-end RIGHT_TAIL

>adaptor1
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
>adaptor2
AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG
>adaptor1_RC
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT
>adaptor2_RC
CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT