Question: Tophat2 Library Type Choice
0
gravatar for Malia
4.6 years ago by
Malia10
University of Utah
Malia10 wrote:

Hello,

I am trying to work out the kinks in my RNAseq alignment pipeline. I am looking at total RNA isolated from Arabidopsis. The ribosomal RNA is removed with the Ribo-Zero kit, and Illumina TruSeq stranded library preparation is performed before 50 bp single-ended sequencing on a Hi-Seq 2000 instrument. 

My question stems from unexpected results gained from the Bowtie2/Tophat2 alignment to the TAIR10 scaffold. According to Illumina and the Tophat manual, I should use the –fr-firststrand argument. However, I ran the alignment three different ways (unstranded, fr-firststrand, fr-secondstrand) and compared the junctions.bed file, as also suggested by the Tophat manual. "The method that produces the most junctions should be the one used."

Unfortunately, the “unstranded” method proved to have the most junctions.

Both fr-firststrand and fr-secondstrand methods yielded the same number of junctions (80023, as seen below).


...
Chr5	26963303	26963527	JUNC00080020	3	-	26963303	26963527	255,0,0	2	32,19	0,205
Chr5	26964985	26965229	JUNC00080021	2	-	26964985	26965229	255,0,0	2	19,32	0,212
Chr5	26968163	26968606	JUNC00080022	14	-	26968163	26968606	255,0,0	2	32,49	0,394
Chr5	26969624	26969961	JUNC00080023	9	-	26969624	26969961	255,0,0	2	46,47	0,290

 

The output from fr-unstranded surprised me, as it also included chloroplast and mitochondrial junctions, unseen in the firststrand and secondstrand output. What was more unusual is that the number of nuclear junctions was higher than the other two methods (81566).


...
Chr5	26964985	26965229	JUNC00081564	2	-	26964985	26965229	255,0,0	2	19,32	0,212
Chr5	26968163	26968606	JUNC00081565	14	-	26968163	26968606	255,0,0	2	32,49	0,394
Chr5	26969624	26969961	JUNC00081566	9	-	26969624	26969961	255,0,0	2	46,47	0,290
...
ChrC	136307	137122	JUNC00081589	1	-	136307	137122	255,0,0	2	21,29	0,786
ChrC	136674	137586	JUNC00081590	39	+	136674	137586	255,0,0	2	49,49	0,863
ChrC	146566	150110	JUNC00081591	1	+	146566	150110	255,0,0	2	40,10	0,3534
ChrM	40535	41977	JUNC00081592	96	+	40535	41977	255,0,0	2	49,49	0,1393
ChrM	42931	59160	JUNC00081593	1	-	42931	59160	255,0,0	2	9,41	0,16188
ChrM	61080	61190	JUNC00081594	1	-	61080	61190	255,0,0	2	28,22	0,88
ChrM	189891	303492	JUNC00081595	2	+	189891	303492	255,0,0	2	19,31	0,113570
ChrM	288059	289051	JUNC00081596	26	+	288059	289051	255,0,0	2	49,49	0,943

So my question: should I go with fr-firststrand as recommended by Illumina?

Or use unstranded since it yields more junctions?

Thanks for your suggestions and advice!

~Malia 

rna-seq library alignment tophat2 • 2.2k views
ADD COMMENTlink written 4.6 years ago by Malia10
1

Its not about how many junction reads you are getting. If you know that the library is strand specific then you should specify it for correct results.

ADD REPLYlink modified 4.6 years ago • written 4.6 years ago by Ashutosh Pandey11k

Oh Sorry Ashutosh Pandey , I just saw that you also had commented similar .

ADD REPLYlink written 4.6 years ago by Manvendra Singh2.1k

No problem Manu. It happens with me too all the time. 

ADD REPLYlink written 4.6 years ago by Ashutosh Pandey11k

Great, thank you all for your help!

ADD REPLYlink written 4.6 years ago by Malia10
1

The method that produces the most junctions should be the one used.

I must say that this is not good advice. What they probably mean is that the right choice usually produces the most alignments but there absolutely no reason to guess. You have the alignment then inspect them with IGV or samtools - it will tell you which reads comes from which file - and that is the answer to what protocol is used.

ADD REPLYlink written 4.6 years ago by Istvan Albert ♦♦ 80k

As you have written above that you sequenced stranded total RNA, then you must specify it. When I was working with Chimeric transcripts from tophat-fusion then many of junctions were false positive.

ADD REPLYlink written 4.6 years ago by Manvendra Singh2.1k
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