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ntremblay 10-09-2018 02:05 PM

AAAAAA.. TTTTTTT.. heterodimers
 
Good evening all,

I have prepared (ds)cDNA from viral RNA extracted from plasma that was subsequently sequenced on a MiSEQ following standard library preparation.

When I got the reads back, I only have TTTTTTTTTTTTTT or AAAAAAAAA sequences for 99.9% of my reads. Read have good phred score and are of the expected length (100 bp).

Any ideas about why I might get such a result?

Thanks!

Nicolas

nucacidhunter 10-09-2018 11:53 PM

Could you post full sequence of few reads (both reads if sequenced paired end).

ntremblay 10-15-2018 10:17 AM

Code:

@m02846:68:000000000-c39d4:1:1101:14422:2079 1:n:0:1
tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt
+
>>afggggegggggggggggggggggggggggfggggggggggggggfffffffffffffffffffffffffffffffffffffffffffffffffffabffffffffffffffffffffffffffffffffffffffffff
@m02846:68:000000000-c39d4:1:1101:14422:2079 2:n:0:1
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
+
bffccecceggggggggggggggcegggggggggggggggggggggggg@?@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@?@??@<@@@@@@@@?@?@@??@@@@@@@@@@@???@@@@@?@@;;;=?;=@@@??@
@m02846:68:000000000-c39d4:1:1101:14218:2110 1:n:0:1
tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt
+
>>aggggfefgcfeggg?ee@egggegcc@@/<afc@@cdgcbf?9bdab;dffffffbffffba=affffaffbbfbfffaafffaf;fffdffffffbd=ddf-9>99b=9@==9-9@-9@bdf;bcff@dcbffa-;9=
M02846:68:000000000-C39D4:1:1101:14218:2110 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
BFAGGCEEEEGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@??@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@;--@->-@99@;@
@M02846:68:000000000-C39D4:1:1101:13886:2230 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>>AGGGGGGGGEEFGGGGFE/@EEGGCCCDGGG?@DCGFFGGB??DGFFFFFF@D>DFFFAA;@FFDABFFAFA@DADDFFFFFFFFA=FBFAABCF;BDDDC-B;-B;FD;>-@FCAFAD--9@BDFAFFFFAFC-@D=@D
@M02846:68:000000000-C39D4:1:1101:13886:2230 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
DFFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@?@@@?@@@@
@M02846:68:000000000-C39D4:1:1101:12194:2260 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>>AFGGGGGGGGGGGCGGGGGGGGGGGGGGGGGGGFFGFGGGGGGGGFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAAFFFFFA=DFFFA;FF;AFFFFFFFFFFFAFF;;ADAFFAFCFFFFAC;C
@M02846:68:000000000-C39D4:1:1101:12194:2260 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
CFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@
@M02846:68:000000000-C39D4:1:1101:12722:2310 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>>AFGEEGAEGE0EFCEEEE@EG/EECCGGGCCCCCC@DCC@@BB@B?BBBBB;@B?;BB==BBBBBBBB?BBBBBBBB?B-9@B?B?@BBB?B?B?B;>BB@B>;-;BBB99B-B;BB--9@-99-B-;B--@;=>@;--@
@M02846:68:000000000-C39D4:1:1101:12722:2310 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
BFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@?@@@@@@@@@@@@@@@@@@=@@@@@@@@@@@?@?@@@@@@@@@??@@@@?@@@@@@@@@@@@@@@@@@?@@@@?@@@@@@@?@@@
@M02846:68:000000000-C39D4:1:1101:20711:2313 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>>AGGGGGGGGGGGGGGGGGGGCFGFGGGGCCGGGGGGGGGGGGGGGFAFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFCAFFFFFFCFFFFFFFFFFFFFFFFFFAFFFFFAFFFFFF
@M02846:68:000000000-C39D4:1:1101:20711:2313 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
CFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@
@M02846:68:000000000-C39D4:1:1101:17123:2486 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTGTTTTGGTGGGGTTTTTTGTGACTTCGACACTGTTT
+
>BBGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFDFA=D----.0000://.-<..--.-...--</00000....-:0000
@M02846:68:000000000-C39D4:1:1101:17123:2486 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
BFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@<=@@@?@@?@@?@?@@?@;??-9@@;=->=@?;?=@<=;-9=@@-
@M02846:68:000000000-C39D4:1:1101:19948:2714 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATAATTTTAATATTTTCGGGTGGGCTATACAACTGCCTTTTTCCTTCTTTTCCT
+
>>AFFGGGGGCGGGG?GCEFGFGGGGGGGFGGGGGGGGGGCG@@@GG<FFFFFFFFFFFFFFFFFFFFFFFDFFFFFAFFFFAF---9.////9///////;///--..---.;.///////;/////;/9/://;/////;
@M02846:68:000000000-C39D4:1:1101:19948:2714 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACAAAAAAACAAAAAAAAAAAAAAAAAAAAAAATAAAAAAAAACAAACC
+
FFFGFGGGGGGGGGGGGEGGGGGGGGGGGGGGGGCGGGGGGGGGGAGAG@@@@@@@@@@@@?@@@@@@@@@@??@?@@@@=?@?@-@-9@?<@@--9A--;@-9-;A-=---9-99;@---9-9-;-;////----------
@M02846:68:000000000-C39D4:1:1101:14946:3025 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>AAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@M02846:68:000000000-C39D4:1:1101:14946:3025 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
CFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
@M02846:68:000000000-C39D4:1:1101:15839:3063 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
3>>FFGCEGGGGEGG?EEGCGCGGEECC?C@CCGGGGGGGGGGGGG@BBBB??B<BBBBBBB?;?BBBB?BBBBBB?BB-@BBBBB?B<BBBBBBBB-@;BB?BB;B??BB?>=?;B@B-BB-@??BBBBB?--BB?-BBB?
@M02846:68:000000000-C39D4:1:1101:15839:3063 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
BFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@?@@@@@@@@
@M02846:68:000000000-C39D4:1:1101:10519:3107 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>>AGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF;BFFFFFFFFAFAFFFF
@M02846:68:000000000-C39D4:1:1101:10519:3107 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
BFFGGGEGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@?@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@?@@@@@@@@@@@@@@@@@?@@@@@@@?@?@@@@@?@@@@
@M02846:68:000000000-C39D4:1:1101:14081:3115 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>>AGGGGGGGEGGGGGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFAFFFFADFFFFFFFFFFFFFFFFFAFFFAFFFFFFFFFABFAFAFFFFFAF;DFFFFFFFAFAFFFAFFFAFFFFFFFFFAFFAFFAACFFACC
@M02846:68:000000000-C39D4:1:1101:14081:3115 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
BFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
@M02846:68:000000000-C39D4:1:1101:10255:3290 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>>AFFGGFGGGFGGFGGGGCGGGGGGGGGGGGGGGGGGGGCG@GGGGFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFAFFFACFFFFFFFFFAFFF
@M02846:68:000000000-C39D4:1:1101:10255:3290 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
CFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
@M02846:68:000000000-C39D4:1:1101:20826:3326 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>AAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@M02846:68:000000000-C39D4:1:1101:20826:3326 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
CFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
@M02846:68:000000000-C39D4:1:1101:9032:3354 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>>AGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFAFFFFFFFAFFFFFFFFFFFFFFFFCAFF
@M02846:68:000000000-C39D4:1:1101:9032:3354 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
CFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
@M02846:68:000000000-C39D4:1:1101:21178:3402 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
>>AFGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFFFFFFFFFFFFFFFFFFFFFFFAFFFFFFFFFFFFFFFFFFFFAAFFFFFFFFFFF;DACFFFFFFFFFFF-DFFFFAAFFAFFFFFFFFFBFF
@M02846:68:000000000-C39D4:1:1101:21178:3402 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
BFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@@@@@@@@@@@@@@
@M02846:68:000000000-C39D4:1:1101:18043:3408 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
3>AEGGGGGGGGGGGGGGGGGGGCGGGGGGGGGGGGGGGGGGGGGGGFFFFFFFFFFF-BBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFCFAA;BFFFFFFFFFFFFFFFFFFFFFFAAB;@B--;--;;-;
@M02846:68:000000000-C39D4:1:1101:18043:3408 2:N:0:1
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
BFAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@?@@@?@@@@@@?@?@@@@@@@@@@
@M02846:68:000000000-C39D4:1:1101:12925:3455 1:N:0:1
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTATACTTAATTTCTTTGAGTGTCTGAGTAATGTCTGACACAGCCACCTTATTATT
+
>>AFFEEEGGGGGGGGEGEEGGCGGCCGC@C@CCCGC@DCCDGGG@GBBBBBB?BBBBB??<BB-@?BBBBB?BB?B;B=BB;B->-9/////////////////.;///9//;///////////9/...9....///;9/9


nucacidhunter 10-16-2018 01:02 AM

I do not know how the library has been prepped but I notice following:

1- It is stranded (directional) library
2- Probably RNA has been poly adenylated after extraction
3- It contains some short fragments (below 100 base)
4- RT reaction probably has been primed with polyT primer

It is library prep issue and not the sequencing. If you would like help you need to give more info on extraction and library prep.

ntremblay 10-16-2018 07:55 AM

Viral RNA was extracted from plasma samples using QIAamp Viral RNA Mini Kit (720uL of sample) using standard protocol.

Double stranded cDNA synthesis was done using Maxima H Minus Double-Stranded cDNA Synthesis Kit (random hexamers, Oligo (dT) and HCV specific pan genomic primers dA20) following standard protocol cycling conditions.

The dsCDNA was quantified by nanodrop and promega QuantiFluorŽ dsDNA System.

I will inquired how the cDNA was prep for sequencing.

Thanks,

Nicolas

nucacidhunter 10-17-2018 12:32 AM

I do not know why oligo dT has been used in 1st strand synthesis. It is possible that dA20 oligo has hybridized to oligo T resulting in creating long stretches of T by joining that has been used as template by reverse transcriptase. Because of high primer concentration polyT artefact has been dominant species in the reactions and has ended up in final library.


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