Having made a paired-end and stranded-forward sequencing (NGS) implies that I will only get positive reading frames?

Question

[sofiablancoglez]

Hello!

I'm doing a de novo assembly with reads obtained with Stranded type illumina sequencing. I guess it's Stranded Forward since the Read 1 map with the cDNA antisense and the Read 2 with the cDNA sense (and therefore they are in the opposite direction, Read 1 sense, Read 2 antisense). However, after doing the assembly de novo, I translate the transcripts obtained and the proteins present a negative frame. Wouldn't it be expected that all of them would have a positive frame since it is a stranded-forward protocol, where the reads sense would be taken as reference? Or am I getting confused and is it really a reverse protocol?

Greetings

[colindaven]

For de novo assembly, if not already completed, try Trinity.

Then you can use the tool transdecoder on your contigs to predict ORFs and get protein seqs etc.

Answer 1

[Istvan Albert]

There are different protocols and the naming is unfortunately often confusing. What you describe is the most common protocol where the second in pair is in sense orientation.

Of course the assembler needs to be aware of the strandedness as well (and should be used with the appropriate parameters) and the actual meaning of the parameters is quite confusing, for example for Stringtie should I use -rf or -fr with the Illumina Trueseq, I always need to look it up and even then I am not sure that I got it right.

Long story short this sounds it was used the wrong way.

Answer 2

[sofiablancoglez]

Thank you very much for your answer! I detected the error. The sequencing protocol was Forward type and implies that the reads (the readings of the mould strings) taken as a reference for the assembly go in a 5'--3' direction, but these are complementary to the 'real' or mould strings and therefore the assembly results in 3'--5' transcripts (real direction of the sequences that were sequenced obtaining reads in opposite directions). Therefore when translated into proteins all the frames are negative.

The truth is that this is an unfortunate tongue twister...