RNA Seq Workflow

Question

[jadepinket]

I am using the RNA-seq workflow: gene-level exploratory analysis and differential expression. I have summerizeoverlap output 0

so <- summarizeOverlaps(ebg, "Epigenomics02-96.alignments.bam", mode="Union",
+                         singleEnd=FALSE,
+                         ignore.strand=TRUE,
+                         fragments=TRUE )
There were 26 warnings (use warnings() to see them)
> warnings()
Warning messages:
1: In .Seqinfo.mergexy(x, y) :
  The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)
2: In .Seqinfo.mergexy(x, y) :
  The 2 combined objects have no sequence levels in common. (Use
  suppressWarnings() to suppress this warning.)

input files Homo_sapiens.GRCh38.89.gtf 02-96.alignments.bam Do I need to sort the bam file Here is how it looks

NB501343:44:HNMWTBGXY:3:21409:23096:14722       0       chrM    713     50      25M1D49M        *       0       0   EEEEEEEEEEEEEEEEEEEEEAEEEEEEEEE/E       AS:i:-8 XM:i:0  XO:i:1  XG:i:1  MD:Z:25^C49     NM:i:1  NH:i:1  XS:A:-  RG:Z
NB501343:44:HNMWTBGXY:1:13201:1233:3457 0       chrM    924     50      24M1I47M        *       0       0       CAATE6EEEEE6EEEE//EEEEEEEEE AS:i:-8 XM:i:0  XO:i:1  XG:i:1  MD:Z:71 NM:i:1  NH:i:1  XS:A:-  RG:Z:0
NB501343:44:HNMWTBGXY:1:13202:26691:3452        0       chrM    924     50      24M1I47M        *       0       0   EEEEEEEEEEEEEEEEEAEE/AAEEEAEEEE AS:i:-8 XM:i:0  XO:i:1  XG:i:1  MD:Z:71 NM:i:1  NH:i:1  XS:A:-  RG:Z:0

Thanks in advance

[cpad0112]

The 2 combined objects have no sequence levels in common.

[jadepinket]

Thanks Devon, I am getting the GTF file from UCSC, I have chosen track: ALL GENCODE V26. Does that seem correct?

[ptinto]

[polite advice] @jadepinket, this is not an answer but a comment, so you should add it as a comment under Devon's answer.

[Devon Ryan]

Post the first few lines of your GTF file, my guess is that somehow you have chromosome names like 1 and MT.

Answer 1

[ptinto]

This seems a classic clash between Ensembl and UCSC coordinates.

I don't know how you have downloaded your gtf file from ucsc, but the "89" part, is the typical notation for Ensembl version Homo_sapiens.GRCh38.89.gtf. Indeed you can download this file from Ensembl here ftp://ftp.ensembl.org/pub/release-89/gtf/homo_sapiens/

UCSC has all chromosome name starting with "chr" and ensembl have only the number. Also the mitochondrial genome in UCSC is chrM and in Ensembl is MT. So your coordinate names from both files are totally different

Answer 2

[Devon Ryan]

Your BAM file uses UCSC chromosome names, your GTF file doesn't.