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08-01-2013, 03:31 AM   #21
kmcarr
Senior Member

Location: USA, Midwest

Join Date: May 2008
Posts: 1,154

Quote:
 Originally Posted by jp. Hiseq2000: PE Read Length: 101 x 2 Insert Size: 80~380 (main 150) Adapter 5': (1).TruSeq Universal Adapter, 58bp; (2.)TruSeq Adapter Index 1-12, 63bp; (3).TruSeq Adapter Index 13-27, 65bp. Q1. Which is the correct -r calculated above of my sample, if any? Is it 250 (+/-14)? Q2. Do I need more information from seq-company to calculate these values ? Q3. What am I missing for calculating insert size ?
--mate-inner-dist is calculated by (mean insert size) - (total read length) so for your data:

150 - (101 x 2) = -52

What this means in biological terms is that, on average, your read pairs overlap by ~52 bp at their 3' ends.

08-01-2013, 06:53 PM   #22
jp.
Senior Member

Location: NikoNarita.jp

Join Date: Jul 2013
Posts: 142

I think, I am getting your point completely with my poor understanding. Here is what tophat says:
-r/--mate-inner-dist <int> This is the expected (mean) inner distance between mate pairs. For, example, for paired end runs with fragments selected at 300bp, where each end is 50bp, you should set -r to be 200. The default is 50bp.
This possibly means:
---50--->|-------200-------|<---50---
= 300 - (50 x 2) = 200
As per your example: - (total read length) so for your data: 150 - (101 x 2) = -52 [What this means in biological terms is that, on average, your read pairs overlap by ~52 bp at their 3' ends].

? Should I give -r -50 ? I think its no problem giving negative value of -r or is there something missing ?

? What about the --mate-std-dev in my case [Read Length: 101 x 2; Insert Size: 80~380 (main 150); Adapter 5': (1).TruSeq Universal Adapter, 58bp; (2.)TruSeq Adapter Index 1-12, 63bp; (3).TruSeq Adapter Index 13-27, 65bp.].
Will it be -120 to 160 ?; if I calculate [(80-380)mean150]: 80 - (101 x 2)= -122 | 380 -(101 x 2) = 178

Am I doing something wrong ?

Quote:
 Originally Posted by kmcarr --mate-inner-dist is calculated by (mean insert size) - (total read length) so for your data: 150 - (101 x 2) = -52 What this means in biological terms is that, on average, your read pairs overlap by ~52 bp at their 3' ends.

Last edited by jp.; 08-01-2013 at 08:35 PM. Reason: adding info

 07-12-2018, 05:16 AM #23 Martin Kanyeki Junior Member   Location: Nairobi Join Date: Jan 2017 Posts: 1 My fragments size on Agarose gel after restriction digestion and ligation range from 200-400bp, the sequencing technology that i use is customized to sequence 80 bases for 80 cycles single read sequencing. Once i trim of the adapter i am left with 75bp from which my SNP markers are scored. ***************Questions*********** Is there any chance that i may be missing some markers since my fragment size was much longer than my read length?