Alignment verification IGV

[ibf123]

Hello folks,

Fairly new to RNA-Seq so here are some quality control questions I've come across and cannot seem to find a good answer to.

After I quality control check my samples with FastQC and proceed with Hisat and samtools, I then check the alignments made using IGV.

However, I've noticed that some of the genes are out by roughly 10-30bp from the reference genome (human 38). Is that ok?

Secondly, if reads map to very few bps for a gene is that gene still a credible hit?

And lastly, is there any concern about having identical mappings of individual genes between different samples?

Thank you.

[ashishbansal]

To view aligned reads using the*Integrative Genomics Viewer (IGV), the SAM or BAM file must be coordinate-sorted and indexed.
1. Always load the reference genome first. Go to*Genomes>Load Genome From Server*or load from the drop-down menu in the upper left corner. Select*Human (1kg, b37+decoy).
2. Load the data file. Go to*File>Load from File*and select*6491_snippet.bam. IGV automatically uses the corresponding*6491_snippet.bai*index in the same folder.
3. Zoom in to see alignments. For our tutorial data, copy and paste*10:96,867,400-96,869,400*into the textbox at the top and press*Go. A 2 kbp region of chromosome 10 comes into view as shown in the screenshot above.
Alongside read data, IGV automatically generates a coverage track that sums the depth of reads for each genomic position.
4. Right-click on the alignment track and*Select by name. Copy and paste*H0164ALXX140820:2:2107:7323:30703*into the read name textbox and press*OK. IGV will highlight two reads corresponding to this query name in bold red.
5. Right-click on the alignment track and select*View as pairs. The two highlighted reads will display in the same row connected by a line as shown in the screenshot.
Because IGV holds in memory a limited set of data overlapping with the genomic interval in view (this is what makes IGV fast), the*select by name*feature also applies only to the data that you call into view. For example, we know this read has a secondary alignment on contig hs37d5 (hs37d5:10,198,000-10,200,000).