Per sequence quality scores

Number of reads: 15462

Mapping

Command name	Command function
/home/students/y06/anastaisha_w/hisat2-2.0.5/hisat2-build chr10.fasta chr10	reference indexing
/home/students/y06/anastaisha_w/hisat2-2.0.5/hisat2 -x chr10 -U chr10.1_out.fastq --no-softclip > chr10.1.sam	creating the alignment in .sam extension, no soft-clipped. chr10 - index file, chr10.1_out.fastq - input fastqc file
samtools view chr10.1.sam -b > chr10.1.bam	converting the alignment to .bam extension (binary file)
samtools sort chr10.1.bam -T temp.txt -o chr10.1_sorted.bam	sotring the .bam file, temp.txt - temporary file, chr10.1_sorted.bam - binary file
samtools index chr10.1_sorted.bam	indexing sorted .bam file

Alignment result

15462 reads; of these:
  15462 (100.00%) were unpaired; of these:
    206 (1.33%) aligned 0 times
    15194 (98.27%) aligned exactly 1 time
    62 (0.40%) aligned >1 times
98.67% overall alignment rate

Here we can see, that 206 reads didn't map on the chromosome, 15257 reads were map.

Counting reads

htseq-count option	htseq-count function
htseq-count -f	specifying the input format
htseq-count -s	specifying, whether the data is from a strand-specific assay.
htseq-count -i	specifying the attributee to be used as a feature ID
htseq-count -m	specifying the htseq-count mode
htseq-count -f bam -s no chr10.1_sorted.bam gencode.v19.chr_patch_hapl_scaff.annotation.gtf > final.txt	getting the htseq result

After using the htseq-count with defaults and -f bam, we can see, that only one gene is presented in the output, meaning that all of the reads are placed to that gene.

braaist@kodomo:/nfs/srv/databases/ngs/braaist$ grep -wv 0 final.txt
ENSG00000165732.8       14643
ENSG00000266122.1       3
__no_feature    610
__not_aligned   206

That was a gene of DExD-box helicase 21 protein with AC ENSG00000165732.8, according to Ensembl DB. Genes of that family are implicated in a number of cellular processes involving alteration of RNA secondary structure such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. Based on their distribution patterns, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division.
To validate, whether all of the 11 reads are placed exactly in the gene borders, it was decided to run htseq-count with the intersection-strict mode. That run could check the hypothesis, because, if the read mapped on a gene partly, that run would give a no-feature result (according to a picture presented below). And it seems that all of the 11 reads were mapped exactly into gene coordinates.

htseq-count modes